Post on 25-Sep-2018
Identification and characterization of novel substrates/interacting partners for the protein tyrosine phosphatase PRL-2
Nau Nau Wong Goodman Cancer Center and the Department of
Biochemistry, McGill University. Montreal
July 2011 A thesis submitted to McGill University in partial fulfillment
of the requirements of the Master degree
© Nau Nau Wong, 2011
Table of Contents Pages
Abstract 1 Résumé 2-3 Acknowledgements 4
Chapter I: Introduction and background 5-25 Phosphorylation and phosphatase 4 PRLs family 7 PRLs structure 8 PRLs phosphatase activity 12 Involvement of PRLs in disease malignance 13 Regulation of PRLs 16 Identified PRLs interacting partners and substrates 18 Cellular pathway regulates by the PRLs family 21 PRL inhibitors 24 Project goals 25
Chapter II: Methods and materials 26-33 Cell culture and transfection 26 Stable transfection 26 Affinity Purification of Mass Spectrometry (AP-MS) 26 Silver-staining 29 Plasmids construction 29 Immunoprecipitation and pull down 30 PCR mouse genotyping 32 Western blot of thymus tissues 32 Murine embryonic fibroblasts (MEF) 33
Chapter III: Results 34-57 Identification of PRL-2 interacting partners/substrates 34 CNNM family members interact with PRL-2 41 PRL-1,2,3 all interact with CNNM3 42 Endogenous interaction of PRL-2 and CNNM3 in several cell 43 lines CNNM3 ACD domain is required for interaction 44 All PRLs interact with CNNM3 via the CBS domains 47 Global phosphorylation did no promoted CNNM3 interaction 52 with PRL-2CSDA trapping mutant PRLs mutations disrupting their interaction with CNNM3 53
PRL-2 knockout mouse model 54 Immortalize MEF cell line 56 Reduced body weight of knockout PRL-2 mouse 57
Chapter IV: Discussion 58-71 Identification of PRL-2 interacting partners using AP-MS 58 CNNM3 interaction with PRL-2 and PRL family 61 PRL-2 binds to CNNM3 at its CBS domains 62 PRL-2 catalytic domain mutations disrupts CNNM3 interaction 68 Model for CNNM3 PRL-2 interaction 69 Physiologic role of PRL-2 71
Conclusion 73
References 74-86
List of Figures
Figure 1: Classification of phosphatase family 6 Figure 2: PRL family 7 Figure 3: Conserved structural domain of PRLs 9 Figure 4: Crystal structure of PRL-1,3 11 Figure 5: Schematic of the AP-MS experiment 28 Figure 6: HEK293 stable cell lines 35 Figure 7: Western blot analysis evaluating the IP efficiency 35 Figure 8: Silver-staining of eluted protein from flag IP 36 Figure 9: CNNM3 interaction with PRL-2 43 Figure 10: PRL-1,2,3 interaction with CNNM3 43 Figure 11: Endogenous interaction of PRL2 and CNNM3 44 Figure 12: Schematic of ACDP structure domains 45 Figure 13: ACD and both CBS domains are important for PRL-2 46
binding Figure 14: PRLs all interact with CNNM3 at its CBS domains 47 Figure 15: Point mutations within the CNNM3 CBS domains 50 Figure 16: D396A and G433D mutation in CBS domains 50
abolished interaction with PRL-2 Figure 17: G433D mutation in full length CNNM3 abolished 52
interaction with PRL-2 Figure 18: increase in global phosphorylation level did not 53 prompt PRL-2 CNNM3 interaction Figure 19: PRL2 C110S and D96A mutation decrease PRL-2 54
interaction with CNNM3 Figure 20: Schematic of gene trap mouse and its PCR genotype 55 Figure 21: Western blot of PRL-2 KO mouse thymus tissues 56 Figure 22: Western blot of PRL-2 KO mouse MEF cell line 57 Figure 23: Body weight of mice measured at 4 weeks time 57
point Figure 24: ACDP family 61 Figure 25: Alignment of CBS domain of CNNM3 with other CBS 67 containing proteins Figure 26: PRL-2 mutation used in this study 69
Tables Table 1: Previously published PRLs substrates and interacting 20 partners Table 2: Primer used in the study 31 Table 3: Non-specific interacting proteins identified in AP-MS 38 Table 4: Potential interacting partners of PRL-2 40 Table 5: Potential interacting partners of PRL-2 CS/DA mutant 40 Table 6: PRLs binding partners identified by large-scale mass 41 spectrometry mapping of human protein-protein interactions Table 7: LC MS/MS peptides unique to each CNNM family 42 Table 8: CBS mutations identified in different CBS domains 49
containing proteins Table 9: CBS domain containing proteins and effect of mutation 64
within their CBS domain
List of Abbreviation
AP-MS Affinity Purification Mass Spectrometry AML Acute myeloid leukemia co-IP Co-immunoprecipitation CRC colorectal carcinoma CNNMs Ancient Conserved Domain Protein Family CRC Colorectal carcinoma DSPs Dual specificity phosphatases EGR-1 Early growth response EMT Epithelial-mesenchymal transition ERK1/2 Extracellular signal-regulated protein kinase ½ Flag beads Flag-M2-Agarose (Sigma)
FTase Farnesyltransferase
GST Glutathione-s-transferase IP Immunoprecipitation KO Knockout Mouse model MKPs Mitogen-activated protein kinase phosphatase MMPs Matrix metalloproteinases NLS nuclear localization signal NCL Neuronal Ceroid Lipofuscinoses PD Pull down PRLs Phosphatase of Regenerating Family PRL1 Phosphatase of regenerating liver 1 PRL2 Phosphatase of regenerating liver 2 PRL3 Phosphatase of regenerating liver 3 PTKs Kinases PPs Protein Phosphatases PTEN Phosphatase and tensin homologue deleted on chromosome 10 PTPs protein tyrosine phosphatases TCL Total cell lysate TGFβ Transforming growth factor beta
1
Abstract
The reversible process of protein phosphosphorylation by kinases and
phosphatases regulates essentially every aspect of cellular processes.
PRLs (Phospshatase of Regenerating Liver) are dual specificity
phosphatases, belonging to the protein tyrosine phosphatase family. PRL
family members (PRL-1, PRL-2 and PRL-3) possess several oncogenic
properties and play an important role in tumoriogenesis and metastasis. In
previous studies using multiple cellular and in vivo models, we confirmed
the role of PRL-2 in cell migration and in the transformation process of
breast cancer. Thus, we seek to identify the cellular pathway, mechanism
of actions, and the physiological function of PRL-2. One of the most
common approaches to elucidating the function of a protein is by
identifying its substrates and/or interacting partners. In this study, we have
identified several interesting PRL-2 interacting proteins using Affinity-
Purification Mass Spectrometry (AP-MS) and we characterized the
interaction with one of these candidates: cyclin M3 (CNNM3). We also
generated a PRL-2 KO mouse models: these PRL2-KO mouse showed
significant weight loss, suggesting PRL-2 might play an essential
physiological role. We believe that the identification of PRL-2 interacting
partners will shed light on the physiological functions of this PTP, and may
lead to the development of new targets for breast cancer therapy.
2
Résumé
L’état de phosphorylation des protéines dans la cellule est
essentiellement régulé par les kinases et les phosphatases de façon
réversible. Les PRLs (Phosphatases of Regenerating Liver) sont des
phosphatases à double spécificité appartenant à la famille des protéines
tyrosine phosphatase. La surexpression des membres de la famille PRL
(PRL-1, PRL-2 et PRL-3) est observée dans une grande variété de
cancers. Ceux-ci possèdent de nombreuses propriétés oncogéniques et
jouent un rôle important au niveau de la génération des tumeurs ainsi que
leur dissémination métastasique. PRL-2 est la moins caractérisée de la
famille PRLs. Nos études effectuées à partir de différentes lignées
cellulaires ainsi que modèles in vivo ont démontrées le rôle de PRL-2
dans la migration cellulaire et le développement de tumeurs du sein. Mes
recherches portent sur l’identification des voies de signalisation cellulaire
modulées par PRL2 ainsi que son mécanisme d’action et son rôle
physiologique. L’approche la plus commune pour comprendre la fonction
d’une protéine est d’identifier ses substrats ou partenaire d’interaction.
Dans cette étude, nous avons identifié plusieurs protéines interagissant
avec PRL-2 in vivo par spectrométrie de masse (MS) et nous avons
caractérisé son interaction avec l’un des candidats de MS : CNNM3. Nous
avons aussi généré un modèle de souris knock-out (KO) de PRL-2. La
souris KO manifeste une perte importante de poids suggérant un rôle
physiologique important de PRL-2. Nous sommes persuadés que
3
l’identification des substrats physiologiques de PRL2 permettra de mieux
comprendre cette PTP et ainsi assurer le développement de nouvelles
cibles afin de fournir un meilleur traitement contre le cancer du sein.
4
Acknowledgement:
I want to offer my sincerest thanks to my great supervisor Dr. Michel
Tremblay for accepting me in his lab and offering me one of the my
greatest learning experiences. My two years of stay in Montreal were full
of joy, not only because Montreal is a great city to live in, but also because
all of the amazing people that I met. I want to thank Dr. Hardy, for he is
always there to lend a helping brain and hand. I have learned so much
from him and he is one of the greatest people I have had the pleasure of
working with. I also want to thank Ailsa for all of her supports; she
supported me through many difficult times; helping me escape from my
evil landlord, feeding me and offering me a words of kindness when my
research hit a rough patch. I want to thank my sister and mother (knowing
that someday in boredom they may might decide to read my thesis). I
thank my mother for letting me to decide what I wanted to do in my life,
although she is clueless as to what science is. I want to thank my sister for
all of her motivational speeches (= verbal abuse), without them I would not
have worked half as hard as I did. I want to thank all of the people in my
karate class, their selfless sacrifice to help me release my stress from
work (I am sorry for all of the bruises!). I want to thank my future children
(in case they feel left out), the thought that someday I might have to make
enough money to feed them motivates me to work hard. Last but not least,
I also want to thank my friend Vanda McNiven, who is always there for
me, to correct my grammar and listen to all my complaints. I want to thank
everyone in the lab, you guys are amazing!
5
Chapter I: Introduction and Background
Phosphorylation and phosphatases
Protein phosphorylation regulates nearly all aspects of cellular physiology,
including metabolism, survival/apoptosis, signal transduction, and cell-cell
interactions (26). This major post-translational modification is a reversible
and precisely orchestrated event involving two opposing groups of
proteins: the kinases and phosphatases (69). Deregulation in this
elaborate signal transduction network results in many diseases, including
diabetes, obesity, osteoporosis, neurodegenerative diseases and various
types of carcinomas (82). Early studies viewed kinases as the “on”
switches while phosphatases were viewed as “off” switches because of
their roles in tumorigenesis: kinases such as Ras and Src were
characterized as oncogenes, while phosphatases such as PTEN
(phosphatase and tensin homologue deleted on chromosome 10) were
characterized as tumor suppressors (13)(87).
Recent discoveries recognize an exchangeable role of kinases and
phosphatases: their functions in positive or negative regulation are
dependent on the effect of phosphorylation on the substrate and its
downstream signalling transduction pathways (93). Some phosphatases
can even target and activate kinases by dephosphorylation, suggesting
that phosphatase activities are just as important as their counterparts in
the regulation of signaling pathways (108). Protein phosphatases (PPs)
are grouped into two major families based on their structure: the Ser/Thr
phosphatases and protein tyrosine phosphatases (PTPs) (108). The
6
human genome contains 107 PTP genes. The amino acid sequence of the
PTP family members are divergent, but they all share a conserved
catalytic motif [(I/V) HCxAGxxR] (105). PTPs can be further grouped into
Cys-based PTPs (Class I, II and III) and Asp-based phosphatases
depending on the key amino acid involved in the catalysis (65). Type I
cysteine based PTPs make up the largest class and include tyrosine
specific classical PTPs or non-classical dual specificity phosphatases
(DSPs) (65). DSPs are defined by their unique ability to dephosphoryate
at phosphotyrosine and/or phosphoserine and/or phosphothreonine within
a substrate (65).This family of PTP includes 61 members, which are
further categorized into sub-families of phosphatases of regenerating liver
(PRLs), and others such as PTEN and mitogen-activated protein kinase
phosphatase (MKPs). PTEN and MKPs regulate major cascade pathways
and are accountable for various diseases such as cancer (65) (Fig.1).
Figure 1: Classification of protein phosphatase families. Phosphatases of Regenerating Liver (PRLs) belong to the subfamily of dual specific phosphatase (DSPs) within Class I PTPs.
7
PRLs family
The PRLs family includes three members: PRL-1 (PTP4A1/PTPCAAX1),
PRL-2 (or PTP4A2/PTPCAAX2/OV-1) and PRL-3 (PTP4A3) (7). PRL-1
was the first in the family to be identified by its modulated expression in
both rat regenerating liver and insulin treated H35 cells (58). The PRL-1
sequence was cloned and its phosphatase activity established four years
after its recognition as an immediate early gene involved in regenerating
liver (22). Subsequently, mPRL-1 and mPRL-3 were found by searching
expressed sequence tag (EST) databases with the PRL-1 sequence
(105). At the same time, PRL-1 and PRL-2 were also identified separately
in a human breast carcinoma cDNA library as a farnesyltransferase
(FTase) substrate protein (16).
PRLs are among the
smallest phosphatases in the
PTP family, it is only ~20kDa
in size. PRLs are located on
three different chromosomes
in humans: 1, 6 and 8
(103)(Fig. 2). Although PRLs
share low sequence similarity with other DSPs, they are highly conserved
within the family and across species (45). Human PRL-2 shares closer
amino acid identity with PRL-1 (87%) than with PRL-3 (76%) (7) (Fig. 4).
Across species, human PRLs display over 40% sequence identity with
lower eukaryotes and above 70% among mammals (45). For instance,
8
human and mouse PRL-1 and PRL-2 protein sequences are completely
identical, while the PRL-3 sequence is 96% identical (82)(105). The high
degree of conservation of PRLs suggests that they carry out critical
cellular functions.
The distribution of PRLs in various human tissues has been studied
extensively (50)(23)(105). PRL-1 and 2 have nearly ubiquitous expression
in all human tissues, while PRL-3 displays a more restricted expression
pattern (23). All three PRLs are expressed in skeletal muscle at high
levels (47). PRL-1 has its highest expression in the brain, PRL-2 has high
levels of expression in all tissues, and PRL-3 is primarily expressed in
heart and skeletal muscle, and has only low expression in all other tissues
(23). In conclusion, although the global expression profiles of PRL-1 and -
2 expression attest to their function in a basic process that is common to
many tissues and cell types, their distinct expression levels and patterns
suggests a different regulatory mechanism for PRLs (7)(103).
PRLs structure
PRLs are one of the smallest in size in the PTP family; therefore they do
not contain additional domains or sequence motifs other than conserved
PTP catalytic domains and a prenylation motif. PTP catalytic domains
include a WPD loop and signature motif (VHCXAGXXR). The prenylation
motif CAAX is conserved in all PRLs and they are similar to those found
in small GTPases of the Ras family (106)(Fig. 3).
9
Figure 3: Domain structure of the PRLs family.
Prenylation at the CAAX motif is critical for the ability of PRLs to anchor at
the membrane and orient towards the cytoplasm (105). PRLs are the only
human PTPs that undergo prenylation, suggesting that they might have a
unique function compared to other PTPs (26). Another conserved region is
a highly polybasic region that occurs immediately before the CAAX
prenylation motif, which is also crucial for PRLs membrane localization.
Two mechanisms have been proposed to explain its function. First, these
basic residues act as a basic patch that interacts favourably with the acidic
membrane, contributing to membrane localization with the prenylation
motif (84). Second, they are also thought to be a potential nuclear
localization signal (NLS). Interestingly, when PRL-1 is prenylated, the NLS
is masked, which results in membrane localization. In the absence of
prenylation these residues act as nuclear localization signals targeting
PRLs to the nucleus (84).
Crystal structures and NMR assignments have been published for PRL-1
and PRL-3. However, only the secondary structure NMR assignment is
available for PRL-2 (7). In Figure 4, we summarized and applied the
previous published structural data of PRL-1 and PRL-3 to PRL-2 (39)(45)
(65). The secondary structures and overall folding of PRLs are highly
similar to one another: they consist of five β-sheets and six α-helices (7)
10
(45). PRLs have the shallowest catalytic cleft of all known phosphatases,
suggesting that they have a broad range of substrate specificity (45). The
surface area near the active site is important in the recognition of PTP
substrates (84). Despite the expected structural similarity between PRL-1
and PRL-3, several variations are found in the active site region: the P
loop and WPD loop do not align between the two structures (39). PRL-3
has more flexible WPD loops due to an extra proline and glycine residue
following the loop (39). In addition, in PRL-3, the side chains of Cys104
and Arg110 is orientated away from the catalytic pockets, creating an
open conformation, while in PRL-1 they are constricted and oriented
towards the pocket (82).
11
12
PRLs Phosphatase activity
The conserved PTP signature motif in PRLs is the first clue linking PRLs
to phosphatases (22). Other than this motif, PRLs do not share any
significant similarity to any PTP described previously. However, PRLs do
demonstrate phosphatase activities (108). First, PRL-1 was able to
dephosphorylate p-nitrophenyl phosphate substrate and this phosphatase
activity can be inhibited by PTP inhibitor orthovanadate. Second,
consistent with other PTPases, a critical cysteine mutation within the
conserved catalytic domain completely abolished its activity (22).
Although PRLs have low sequence identity with other DSPs (<30%),
they are still classified as a member of DSPs because of their
secondary structure element and overall fold (45). PRLs have the
closest sequence similarity to PTEN and Cdc14, but they have the
closest structure similarity to VHR, PTEN, MKP and KAP (7)(82).
Functionally, it is still not clear if PRLs dephosphorylate at tyrosine
and serine/threonine. Although the tyrosine phosphatase inhibitor
(orthovanadate) inhibits PRL-1, Ser/thr phosphatase inhibitors such
as okadaic acid, sodium fluoride, and calyculin A have no inhibitory
effect (50). In addition, PRLs show strong preference for
phosphotyrosine-containing peptides, whereas they have no activity
with phosphothreonine-containing peptides (22)(102). PRLs
phosphatase activities are essential for enhanced cell growth and cell
transformation, therefore the identification of PRLs substrates is
critical for clarifying its mechanism (46).
13
Although classical PTPs and DSPs dephosphorylate very different
residues, they share the same general catalytic mechanism (45). PRLs
dephosphorylation involves two essential amino acids within two
conserved areas: asparatic acid in the WPD loop and cysteine within the
CX5R catalytic pocket (39)(Figure 3). Dephosphorylation starts with the
insertion of the substrate phospho-amino acid into the catalytic pocket.
Upon substrate docking, the WPD loop adopts a closed conformation and
covers the active site like a “flap” (39). The conserved cysteine
nucleophile attacks the substrate thioester, forming a covalent thio-
phosphate intermediate (13). Aspartic acid in the WPD loop then acts as a
general acid, donating a proton to the leaving phosphate group. The same
aspartic acid then acts as a base to assist the nucleophilic attack by a
water molecule, releasing the substrate and inorganic phosphate (7)(13).
Involvement of PRLs in disease malignance
Particular interest in the PRLs family is generated mostly by its association
with several aspects and types of human carcinomas. Elevated
expression of PRLs is involved in tumor growth, angiogenesis, metastasis
and poor prognosis (31) (70)(99)(104). PRL-3 is the most studied member
of the PRLs due to its key role in mediating the progression and
metastasis of colorectal carcinoma (CRC). PRL-3 expression is virtually
undetectable in normal colon epithelial, but its expression is escalated
from advanced primary tumors to CRC liver metastasis (40). PRL-3 is also
involved in melanoma metastasis (96), breast carcinoma (74), gastric
14
carcinomas (70), liver carcinoma (96), and ovarian cancer (92). PRL-3
expression has been established as an excellent prognostic factor in
predicting the development of liver and lung metastasis (7). Similarly,
PRL-1 overexpression is also found in several cancers, including
melanoma (93), pancreatic cancer (81), lung cancer cell lines (1) and
esophageal squamous cell carcinoma (93). PRL-2 overexpression is
detected in pancreatic cancer (81), prostate cancer (94), acute myeloid
leukemia (AML) (99), and lung cancer (97). PRL-2 is involved in prostate
tumor progression, its expression is up-regulated in both prostate tumor
cells and advanced prostate cancer (94). PRL-2 is also associated with
hematopoietic malignancies in several studies. Overexpression of PRL-2
contributes to poor prognosis of acute myeloid leukemia (AML). Its
chromosomal anomaly is frequently linked to leukemia/lymphoma and it is
upregulated in Hodgkin’s lymphoma cell line (4)(99). We also showed that
PRL-2 expression is elevated in laser capture microdissected primary
breast tumour epithelial cells when compared to matched normal tissues
in our previous study (33). Clearly, all clinical data point to the involvement
of PRLs in cancer progression. PRLs contributes to several hallmarks of
cancers, including increased cell proliferation, migration/invasion,
angiogenesis and the inhibition of apoptosis (40)(95). Aberrations of PRLs
expression was studied in numerous cell lines using soft agar, matrigel
invasion, transwell migration and wound healing assays. Unyielding
results show that overexpression of PRLs altered cell morphology,
increased colony formation, promoted cell growth, migration and wound
15
healing, and enhanced cell adhesion and spread. Conversely, the
knockdown and catalytic dead PRLs mutant reduced the motility and
invasive property (4)(16)(46)(48)(50)(57)(59)(69)(81)(84)(92)(96)(97)
(104). We have also shown PRL-2 overexpression increases colony
formation and cell migration in TM15 and DB7 cells (33). PRL-3
participates in angiogenesis. PRL-3 recruits endothelial cell forming blood
vessels to support bigger tumor cells and it also down-regulates the
secretion of IL-4, a known negative vasculogenesis (31). To date, no
evidence has suggested the involvement of PRL-1 or -2 in angiogenesis.
The physiological role of PRLs in vivo has also been studied in mouse
models. Tail vein injections of several overexpressing PRLs cell lines
promote tumor formation and metastasis in nude mice (16)(96)(104). We
have also demonstrated that injection of PRL-2 overexpressing cells in the
mouse mammary fat pad promotes tumor formation (33). Beside its
oncogenic role, it is not a surprise to find that PRLs are also involved in
other biological processes. PRL-1 is involved in embryonic development,
its expression is elevated during the development of mouse neuronal,
gastrointestinal and skeletal tissue (95). In addition, oxidative stress in
cone photoreceptors increases PRL-1 expression, suggesting an
involvement of PRL-1 in the phototransduction cascade (102). PRL-3
mediates angiotensin II (AngII), a factor that triggers a series of signaling
events leading to cardiac hypertrophy (50). Curiously, PRL-2 expression is
upregulated during hibernation in bats brain tissue, suggesting that PRL-2
maybe involved in maintaining normal cycle of nerve cells in the
16
hibernating bat (101). PRL-2 was also identified in Neuronal Ceroid
Lipofuscinoses (NCLs) mouse models as a protein critical for the neuronal
growth of cone-cytoskeletal dynamics (89).
Regulation of PRLs
PRLs activity is regulated at several levels: transcription,
phosphorylation, localization, oligomerization and oxidation.
PRLs genomic sequences are highly conserved except at the 5’ non-
coding region, suggesting PRL members are differentially controlled at the
transcriptional level (105). Several transcription factors were identified to
regulate PRL expression. PRL-1 and 3 are direct transcriptional targets of
p53 since they both contain a p53 binding site within their promoters (6).
In addition, an increase of p53 expression increased PRL-1 and 3 mRNA
levels in a dosage dependent manner (56). PRL-1 also contains a binding
motif for a growth activated transcription factor known as early growth
response (Egr-1)(82). In NIH3T3 cells, overexpression of Egr-1 increases
PRL-1 gene transcription (68). PRL-3 is a direct target of the TGFβ
signaling pathway; the binding of Smad3/4 to the PRL-3 promoter site
downregulates its transcription (40). Since the TGFβ signal is frequently
lost in colorectal carcinoma, PRL-3 upregulation in CRC is proposed to be
regulated by the TGFβ pathway (40). As of yet PRL-2 transcription
regulation has not been described.
PRLs subcellular localization is controlled by conserved PRLs CAAX
domains and polybasic regions. The farnesylation of the CAAX domain is
17
essential for their subcellular localization to the membrane and the
endosomal compartment. Prenylation deleted mutants (SAAX) and
farnesyl transferase inhibitor (FTI) treatment redistributes PRLs to the
nucleus (13). Also, PRL-1 localizes in a cell cycle dependent manner; in
non-mitotic cells, it localizes to the membrane and endoplasmic reticulum
while in mitotic cells it relocalizes to the centrosome and spindle apparatus
(93). Interestingly, PRLs promote invasion and motility and cell cycle
control in a farnesylation dependent manner. FTI treatment and PRL-1
SAAX mutants induced defects in mitosis and cytokinesis. In addition, they
also thoroughly inhibit the invasion and motility in SW480 cells (26)(93).
Deletion of the polybasic region of PRL-1 not only redistributed the
enzyme from the membrane to the cytoplasm it also abolished cell growth
and migration (84). Together, these results suggest that subcellular
localization of PRLs is important for their functions in regulating cell
growth, invasion and migration. Oxidation regulates the phosphatase
activity of PRL-1 and PRL-3 by of the formation of an intramolecular
disulfide bridge between cys49 and cys104 (45)(102). This disulfide bond
imposes a conformational constraint on the phosphatase active site,
inhibiting catalysis and substrate binding (65). Disulfide bridge formation
was suggested as a mechanism to prevent the permanent inhibition of
PRLs by irreversible oxidation (45). Interestingly, oxidative stress induces
PRL-1 disulfide bridge formation and inactivated phosphatase activity is
observed in mouse retinas stimulated with continuous illumination (45).
Given the conservation of the two cysteines in PRL-1 and 3, we expect
18
that PRL-2 activity could also be modulated by oxidative stress.
Trimerization of PRL-1 is novel among the PTPs (84). Trimerization is
postulated to be essential for PRL function as PRL-1 structure has always
been crystallized in its trimeric form (84). Mutation within the trimeric
interface disrupted PRL-1 trimerization, abolishing the proliferation and
migration phenotype in a phosphatase activity independent manner (39)
Previous studies suggested that the trimerization and membrane
localization cooperate with each other to provide stronger adhering forces
to the membrane (39). In addition, trimerization could also increases the
affinity and substrate specificity of PRL-1 to its substrates (84). The
trimeric interface structure is conserved in all PRLs therefore
oligomerization might be a preserved and common regulatory mechanism
(84).
Identified PRLs interacting partners and substrates
PRLs interacting partners and substrates are involved in diverse aspects
of cellular function (Table 1). PRL-3 interacts with transcription factor ATF-
5 and elongation factor EF2 to regulate protein synthesis (62)(69). PRL-3
also mediates EMT processes with several cell adhesion proteins
(integrins and CDH22) and components of cytoskeleton remodelling
(tubulin, stathmin and keratin). PRL-1 and PRL-3 both interact with α-
tubulin, the major constituent of microtubules (93). PRL-3 also interacts
with stathmin, a regulator of the microtubule dynamic (109). Keratin-8 is a
component of the intermediate filament, and reorganization of keratin
mediates cell migration (57). Phosphorylation regulates the activation of
19
keratin, nucleolin, integrin and Ezrin and PRLs overexpression has been
found to decreases all their phosphorylation levels (27)(57)(67)(78)(97).
PRL-2 has one known interacting partners, geranylgeranyl transferase
(βGTTII). PRL-2 competes with the α-subunit of GGTII for βGGTII binding,
inhibiting the GGTIIα/β subunit association. The GGTIIα/β complex is
important for the prenylation of Rab, therefore PRL-2 is thought to regulate
the vesicle trafficking through Rab mediated protein recycles (79). Erzin is
the first substrate identified for both PRL-2 and PRL-3 (27)(97). PRL-2 and
3 dephosphorylate specific Tyr and Thr sites on Erzin. Erzin is involved in
tumor invasiveness; a previous study showed that PRL-3 mediates its
angiogenesis property through dephosphorylation of Erzin (27).
20
21
Cellular pathway regulated by the PRLs family
Given the role of PRLs in tumorigenesis, it is not surprising to find that
several cellular pathways mediated by the PRL family are related to
cell proliferation, apoptosis and metastasis (31)(104).
PRLs play a critical role in mediating the progression of cells through
mitosis. Overexpression of PRL-1 and -2 promotes a cell population
transition from the G1 to S phase in a p53-dependent pathway (95).
PRL-1 reduces p53 levels by increasing its proteosomal degradation
either directly by the activation of MDM2, or indirectly by the up regulation
of PIRH2 (56). Decreased amounts of p53 diminishes the activity of
downstream p21Cip/Waf1 (p21), a cyclin kinase inhibitors of CDK2. In
time, the enhanced CDK activity allows the cell to bypass the G1
restriction point and entry into S phase (95). PRL-3 knockdown also
inhibits cell proliferation by arresting the cell cycle at the G1 phase. Cell
cycle arrest is regulated by a p53-dependent and a PI3K/AKT dependent
pathway (6). A decrease in PRL-3 level up-regulates p19ART, a MDM2
sequestration protein. A reduction in the amount of MDM2 sequestered
therefore increase p53 stability. In addition, a decrease in PRL-3 activates
the PI3K/AKT pathway and increases phosphorylation of FOXO, a
transcription factor. FOXO then up regulates cell cycle regulatory genes,
such as p21/p27 (6). PRL-2 expression does not alter total p53 levels,
suggesting that PRL-2 may exert its cell cycle control in a p53
independent manner (97).
22
Metastasis is a multi-step process in which cancer cells spread from the
primary tumor to distant locations. Epithelial-mesenchymal transition
(EMT) is an central step in metastasis - it is a process in which cancer
cells gain an increase in cell motility and invasiveness (48)(92).
Overexpressing PRL-1 and 3 cells exhibit an EMT change in cell
morphology (69)(96). Studies later showed that PRL-3 mediates EMT
interference of cell-cell adhesion, focal adhesion complexes and promotes
cytoskeleton rearrangement (48).
Cells adhere to each other by adherences junctions and a large group of
cell adhesion molecules called cadherins. PRL-3 can mediate cell-cell
adhesion either directly by suppressing expression of CDH22/E-cadherin
or indirectly through the PTEN-PI3K-Akt signaling network (47). PRL-3
overexpression up-regulates PTEN/PI3K and activates AKT. phosphoART
phosphorylate and inactivates GSK-3β, therefore increasing Snail activity.
Raised Snail subsequently down-regulates critical components of the
adherence junction complex, such as E-cadherin, y-catenin and
cytokeratin (92).
A focal adhesion is a protein complex that attaches cells to the
extracellular matrix (ECM) (7). PRLs increase cell motility and invasion by
disrupting focal adhesion complexes (49). PRL-3 increases Src activity by
reducing the expression of Src kinase (Csk), a kinase that inhibits Src
acitivity by phosphorylation. Enhanced Src phosphorylation then activates
focal adhesion kinase (FAK) and p130Cas; together, they delay focal
adhesion turnover (46). PRL-3 also directly reduces the number of cellular
23
focal adhesion complexes by down-regulating components of focal
adhesion, such as paxillin and vinculin (92). PRLs increase invasiveness
through common pathways, as they all up-regulate p130Cas (1)(49)(97).
However unlike in PRL-1 and 3 c-Src activity is unchanged in PRL-2. This
suggests that PRL-2 regulates p130Cas activity by an alternative pathway
(49).
PRL-1 and 3 also mediates cytoskeleton rearrangement through Rho
family GTPase, an important regulator of actin cytoskeleton reorganization
(26)(48). PRL-1 and 3 decrease the levels of RhoA GTP and Rac1 GTP,
which results in an increase in cell spreading (1)(92).
Extracellular signal-regulated protein kinase ½ (ERK½) is a major
signaling pathway involved in several fundamental cellular processes (46).
PRLs overexpression increases the phosphorylation level and the activity
of ERK½ activity (48)(67)(33). Activation of ERK½ by PRL-3 drives up
regulation of MMP2 and MMP9 by Ap1 and Sp1 transcription factors (48).
Matrix metalloproteinases (MMPs) are major hydrolytic enzymes that
break up the ECM, increased expression of MMPs allows cell invasion into
the surrounding tissue (48). In our previous study, we showed that PRL-2
overexpressing tumors generated from MMTV PRL-2 mice crossed with
ErB2 mice displayed increase phosphorylation of ERK1/2 (33).
The PRLs family participates in a variety of cellular pathways and some of
which are shared among all PRLs. However, studies to date show PRL-2
is distinct from other PRLs as it is not involved in some major pathways,
such as the PTEN/PI3K/AKT, cSrc and p53 pathways. PRL-2 does not
24
activate p53, Akt and c-Src suggesting that PRL-2 functions mediate a
pathway that is different from PRL-1 and 3 (97). Thus the identification of
PRL-2 substrates will be critical for elucidating its role in signaling
pathways.
PRL inhibitors
PRLs are very attractive targets for anti-cancer therapy because of their
unquestionable involvement in tumorigenesis. Several inhibitors have
been identified to date, including pentamidine, prenylation inhibitor,
rhodanine, natural compound and thienopyridone. Pentamidine was the
first PRLs inhibitor to be identified, it is known to inhibit growth of several
cancer cell lines. However, pentamidine also has an inhibitory effect on
several other PTPs. Therefore, it is not conclusive as to whether the
inhibitory effect is mediated by PRL or another phosphatase family (7).
Given that PRLs are the only members in the PTP family that undergo
prenylation, a lot of interest was placed on the Farnesyltransferase
(FTase) inhibitors (TIFs) and Geranylgeranyltransferase inhibitos
(GGTTs). However, these inhibitors proved ineffective in clinical trials
because they have an effect on a wide spectrum of targets outside of the
PTPs (7). High throughtput screening of chemical libraries discovered
several inhibitors, such as rhodanine and the natural compounds
biflavinoids ginkgetin and sciadopitysin (3). However, their specificity,
toxicity and mechanism of action have not been investigated.
Thienopyridone is a selective small molecule PRL inhibitors and it
suppresses tumor cell anchorage independent growth through p130Case
25
cleavage and anoikis (20). In addition, it also has a good selectivity for
PRLs but not for other PTPs (49). To date, however clinically useful and
specific inhibitors of PRLs have not been reported (64). Given the high
homology between PRL members, it is difficult to identify specific
inhibitors for each PRLs member. PRLs might share similar functions
therefore knockdown of one PRL may be compensated by other PRLs
member. The identification of a PRL-2 downstream substrate might
provide an easier downstream target for inhibitor design and a better
substrate for an in vitro phosphatase assay to evaluate PRL inhibitors
(97).
Project Goals
Although mounting evidence has established the role of PRLs in tumor
progression and metastasis, the mechanism of their regulation and the
basis of their transforming activity are not yet understood. We have
previously identified PRL-2 as a PTP that is overexpressed during breast
cancer development (33). In multiple breast cancer cellular models, we
have confirmed the role of PRL-2 in cell migration and transformation.
Also, PRL-2 promoted tumor formation in vivo in both a xenograft model
and in breast-cancer prone MMTV-ErbB2 transgenic mice. Thus, the
identification of bona fide physiological substrates or interacting partners
of PRL-2 is the first step to understanding its mechanism of action and its
downstream signalling pathway. In addition, we generated a PRL-2
knockout mouse model to provide insight into the physiological function of
PRL-2.
26
Chapter II: Methods and materials
Cell culture and transfection
Cell lines were cultured in DMEM (Hyclone) supplemented with 10%
FBS and 10µg/ml gentamicin at 37°C with 5% CO2. Transfections were
performed using lipofetamine 2000 (Invitrogen) following the
manufacturer’s instructions and media were replaced 4hr after
transfection. Experiments were generally carried out 24hr post-
transfection.
Stable Transfection
HEK293 cells were cultured to 80% confluence and transfected with
pcDNA3.1 Flag vector, Flag-PRL-2 or Flag-PRL-2CSDA vector. To
select for stable transfectants, 200ug/ml of G418 was added to the
medium 48hr after transfection.
Affinity Purification of Mass Spectrometry (AP-MS)
AP-MS was performed as described (17). Five plates (p150) of 80%
confluent stable cells were harvested for each AP-MS. Cells were scraped
and washed in ice cold PBS 3x before the addition of 1mL lysis buffer
(50mM Hepes-KOH, pH8.0, 100mM KCl, 2mM EDTA, 0.1% NP-40, 10%
glycerol and 10mM NaF), freshly supplemented with 1x protease inhibitor
and 1mM DDT. Lysate was incubated on ice for 20 min to facilitate the
lysis process before undergoing two freeze-thaw cycles. Cell debris was
27
cleared by centrifugation at 13,000g for 15min. Lysate concentrations
were measured by Bradford assays and equal amounts of lysate were
incubated with 100ul washed Sepharose beads to reduce non specific
interactions. After 1hr of incubation at 4oC, the beads were pelleted and
removed. 7ul (per 10mg of pre-cleared lysate) of pre-washed packed
FLAG-M2 agarose (Sigma) beads were added to the lysate and together
they rotated with gentle end over end agitation overnight at 4oC. Beads
were washed extensively with lysis buffer and FLAG rinsing buffer (50mM,
NH4HCO3, pH8.0, 75mM KCL and 2mM EDTA), followed by addition of
500ul of freshly prepared elution buffer (0.5M NH4OH, pH~11.0, 0.5mM
EDTA). Eluted products were then lyophilized by speed vacuum, trypsin
digested and analyzed with LC/MS/MS at the McGill Mass Spectrometry
Facility.
28
29
Silver staining
Polyacrylamide gels were fixed in 12% acetic acid, 50% ethanol and
0.05% formaldehyde overnight. The next day, they were washed in 20%
ethanol, sensitized for 2 min in 0.02% sodium thiosulfate, and rinsed with
water twice. Gels were incubated for 20min in the staining solution (0.2%
silver nitrate, 0.076% formaldehyde), rinsed with large volumes of water
and developed in freshly prepared developing solution (6% sodium
carbonate, 0.0004% sodium thiosulfate and 0.05% formaldehyde). The
reaction was stopped by adding 12% acetic acid.
Plasmid construction
CNNM3 fragment was amplified from CNNM3 cDNA (Openbiosystem)
using KAPA HiFi DNA Polymerase (Kappa Biosystem). The PCR mixture
contained a final concentration of 1xKAPA GC HiFi buffer, 2mM dNTP,
50ng of CNNM3 vector, and 0.4ul of Kapa Hifi polymerase. The PCR
program was 95oC for 3mins, followed by 30 cycles of 98oC for 20s, 60oC
for 15s, and 72oC for 2mins. Purified PCR products were cut and inserted
into a pcDNA3 Flag vector. CNNM3 domain truncated mutants cDNA was
amplified by specific primers flanking the sequence of interest (Table 2).
The point deletion mutants of CNNM3 were generated by site
mutagenesis using Kapa Hifi DNA pol. The same PCR mixture as above
was used, except that Flag 2xCBS or Flag CNNM3 vectors were used as
the template. PCR condition changes included increasing the annealing
30
temperature to 60oC for 1min and the extension time to 72oC for 10min.
PCR products were digested overnight with DpnI, and transformed into
DH5α.
Immunoprecipitation and pull down
Cells were lysed with Triton buffer (1% Triton, 20mM Tris and 150mM
NaCl pH7.5) supplemented with protease inhibitor (Sigma), cleared by
centrifuging at 13,000×g for 10mins and quantified using BCA Protein
Assay (Thermoscience). For immunoprecipitation (IP), equal amounts of
proteins (~3mg) were incubated with 2ul of PRL-2 antibody (Milipore) or
CNNM3 antibody (Protecintech) overnight. The next day, 24ul of pre-
washed agaroseA/B bead (Bioshop) were added to the lysate for an
additional 2hr incubation. In the GST pull down assay, approximately
400ug of proteins were incubated with 16ul of pre-washed glutathione
sepharose (GE Healthcare) for 2hr at 4oC. In Flag IP 5ul of M2-Flag resin
(beads) were added and incubated overnight. In all cases, beads were
washed several times with lysis buffer and protein were eluted by boiling
the beads in 2xSDS loading buffer.
31
32
PCR mouse genotyping
Genomic DNA was isolated from mouse tails using standard pheno-
chloroform extraction. Multiplex PCR used three primers F1, R2, and R3
(Table 2) (Fig. 20). The PCR mixture contained a final concentration of 1x
reaction buffer, 2mM dNTP, 1ul DFS Taq Pol (Bioron), 0.4uM of each
primer, and 1ul of ~50ug/ul of purified genomic DNA in a total volume of
20ul. PCR conditions were 95oC for 2mins, followed by 30 cycles of 95oC
for 45s, 60oC for 1 min, and 72oC for 80s.
Western blot of thymus tissues
Thymus tissues were isolated, snap freezed in liquid nitrogen and
stored at -80oC. To prepare protein lysates, thymus tissues were
homogenized in Ripa lysis buffer (150mM NaCl, 1% NP-40, 0.5% sodium
deoxycholate, 0.1% SDS and 50 mM Tris, pH 8.0) with freshly added
complete protease inhibitors (Roche biochemicals) on ice. The
homogenates were centrifuged at 13,000g for 10min at 4oC, and the
concentration quantified by Bradford assay. Equal amounts of cell lysates
were resolved by 12% SDS-polyacrylamide gel electrophoresis (PAGE)
and transferred to PVDF. Membranes were blotted in milk for 1hr followed
by incubation of PRL-2 antibody (Millipore) in PBS-T (Phosphate buffered
saline with 0.1% Tween-20) overnight at 4oC. The next day, membranes
were blotted with anti-mouse secondary antibody (1:10000).
33
Immunoreactive proteins were detected with enhanced
chemiluminescence detection reagents (Western Lightning Plus)
according to the manufacturer’s instructions.
Murine embryonic fibroblasts (MEF)
The PRL-2 KO mouse was generated by gene trapped ES cell (Sanger
Institute). Heterozygote mice were set up for mating, and embryos were
isolated at E14.5. Harvested embryos were washed multiple times in PBS,
and red tissues were removed and finely minced. Each embryo was
digested with 6ml 0.25% Trypsin/EDTA containing 10mM HEPES at 37oC
for 30min. Large chunks of tissues were removed by centrifugation at
1000rpm for 5min. The cells in the supernatants were plated in 150cm
dishes, and incubated at 37oC with 5%CO2.
34
Chapter III: Results
Identification of PRL-2 interacting partners and substrates
Several techniques can be used to identify interacting partners/substrates
of PRLs, including yeast two hybrid (47)(107), affinity purification (78) and
differential protein expression profiling (57)(62). In our study, we combined
these techniques with the PTP trapping system to identify physiological
substrates. The use of trapping mutants increases the likelihood of
isolating substrate complexes as it reduces the transient nature of this
complex seen in wildtype PTPs (46). We have previously attempted
PRL-2 yeast two hybrid experiments. However, we were unable to identify
any potential candidates (data not shown). We switched our strategies to
a FLAG tag affinity purification system as previously described (17)(25). In
order to generate enough protein for affinity purification, we created a pool
of stable HEK293 cell lines overexpressing Flag empty, Flag wildtype, and
Flag PRL-2CSDA substrate trapping mutants. PRL-2CSDA mutants
contains two mutations, D69S and C110S, within the conserved catalytic
domain of PTPs (Fig. 26). They block the ongoing PTP catalysis,
stabilizing the enzyme-substrate interaction, and therefore trapping the
substrate within the PTP catalytic pocket (9). We have successfully
generated a stable HEK293 cell lines expressing moderate amounts of
Flag PRL-2 and PRL-2CSDA (Fig. 6).
35
We performed a large-scale affinity purification and western blot to
validate the efficiency of the IP and protein elution (Fig. 7). Although some
Flag PRL-2 still remains on the beads fraction, we detected flag PRL-2 in
the eluted fraction, validating successful IP and elution of PRL-2 and its
putative interacting partners.
Silver-staining was performed on the eluted proteins to identify the
difference in banding patterns between Flag empty control, Flag PRL-2
and Flag PRL-2CSDA (Fig.8). Several bands appeared in Flag PRL-2 and
PRL-2CSDA, but not in the flag empty, suggesting these are interacting
partners that associate with PRL-2.
36
However, these bands could not be directly identified from the gel by mass
spectrometry because of their low intensity. Therefore we adapted a
technique that combined the affinity purification step directly to mass
spectrometry (AP-MS). In total, three independent AP-MS were performed
under similar conditions. A list of background/non-specific binding present
37
in the IP of flag empty samples are listed in Table 3. In addition, we also
cross referenced them with previously published common background
contaminants using the same technique and cell line (17).
38
39
Table 4 and 5 contain candidate interacting partners/substrates
immunoprecipited using PRL-2 wildtype or PRL-2 CSDA but not present
in the Flag control. In our results, several candidates interacted with both
PRL-2 wildtype and trapping mutant. They include nucleolin, Y box
binding protein 1 (YBX1), translocated in Liposarcoma Protein (TLS), and
PRL-1. Analysis of peptide sequence coverage has isolated peptide
sequences unique to PRL-1, indicating PRL-2 interact with PRL-1 in vivo.
A couple of candidates also interact only with PRL-2CSDA trapping
mutants, including 14-3-3 (14-3-3ε, ζ/σ), casein kinase II (CKII), Gem
associated protein (Gemin3,6), serine/threonine kinase receptor
associated protein (STRAP), SET translocation (SET) and survival of
motor neuron (SMN). Table 6 summarizes results from a large scale
mapping of human protein-protein interactions by mass spectrometry
(25). In this study, the researchers included PRL1,2,3 using similar
methods to look at the interacting partners of human bait proteins (25).
40
41
CNNM family members interact with PRL-2
The Ancient Conserved Domain Protein (ACDP) family consists of 4
members, CNNM1-4 (also referred to as ACPD1-4 or cyclinM1-4). Here
we showed that wildtype PRL-2 pulled down all member(s) of ACDP in
three separate AP-MS experiments (Table 4). Analysis of the peptide
coverage mapped sequences that are unique to each CNNM members
(Table 7). Furthermore, CNNM3 and 4 also interact with PRL-2 and 3 in
the large scale IP (Table 6). The consistent IP results and redundant
interaction with all members of the CNNM family suggests a conserved
and important function of this interaction. However, we decided to focus
42
our interest on CNNM3 because it has been IPed in all AP-MS
experiments.
PRL-1,2,3 all interact with CNNM3
We validated the interaction of PRL-2 with CNNM3 in by a co-transfected
Myc-CNNM3 and Flag PRL-2 in HEK293 cell followed by IP with a Flag
antibody (Fig. 9). In accordance with the AP-MS, PRL-2 interacted with
CNNM3, but the interaction was lost with the PRL-2CSDA trapping
mutant.
43
We want to know if all PRLs interact with CNNM3 since they share high
sequence similarity, similar structure, and possibly functional homology.
Also, previous studies identified common interacting partners between
PRLs (Table 1). We showed in a GST pull down that all members of PRLs
interact with CNNM3 (Fig. 10).
44
Endogenous interaction of PRL-2 and CNNM3 in several cell lines
In order to eliminate the possibility that the interaction is an artefact of the
AP-MS system and/or protein overexpression, we performed a co-
immunoprecipitation (co-IP) experiment in several cell lines. Protein
lysates from different cell lines were immunoprecipitated with PRL-2 and
CNNM3 antibody separately. We observed that not only PRL-2 antibody
IP CNNM3, but reversibly CNNM3 antibody is also able to IP PRL-2 in all
four cell lines, These results confirms the physiological interaction
between endogenous PRL-2 and CNNM3 in vivo (Figure 7).
45
CNNM3 ACD domain is required for interaction
Knowing which CNNM3 functional domain interacts with PRL-2 can give
insight into the mechanism of interaction. All CNNMs possess four
transmembrane domains and an ancient conserved domain (ACD)
domain (Fig. 12).
Figure 12: Schematic of ACDP family conserved structure domains
As the name implies, the ACD domain is highly conserved within ACDP, it
consists of two cystathionine-beta-synthase (CBS) domains, a cyclin box
and a cyclic nucleotide-monophosphate-binding domain (cNMP) binding
domain (91). Six CNNM3 truncated mutants fused to Flag were created
based on the predicted domain structure of CNNM3. A GST PD assay
was performed on cells co-transfected with Flag fused truncated mutants
and GST-PRL-2 (Fig.13). According to an SDS-PAGE of total cell lysis
(TCL), all constructs were expressed at the expected size. Interaction was
lost in the ∆282 mutant that contains just the transmembrane domain
suggesting that the ACD domain is critical in mediating PRL-2 interaction.
∆380 is another mutant that lost the interaction. It contains a
transmembrane domain and one CBS suggesting that both CBS domains
are essential for the interaction.
46
47
All PRLs interact with CNNM3 via the CBS domains
We produced two additional deletion mutants spanning the ACD region:
the 2xCBS included both CBS domains and ccbox composed of the cyclic
motif and cNMP binding domain. PRL-1 and PRL-3 were also included in
the co-IP experiment because we want to know if the interaction occurs at
the same domain for all the PRLs. Flag IP is performed on Flag
2xCBS/ccbox and its interaction with GST-PRL-1,2,3 (Fig. 14). PRL-1,2,3
bind to the 2xCBS domain, but not the cyclin box and cNMP binding
domain.
48
We have demonstrated in ACD domain that CBS domains alone are
sufficient for PRLs interaction and that this binding shared a common
region within the PRL family.
Point mutations within the CBS domain disrupted PRL-2 binding
CBS domains are found in a variety of proteins with diverse functions
and mutations within these CBS domain are associated with several
inheritable diseases (8)(76). Lists of pathogenic CBS domain mutations
were generated from an extensive literature search and we performed
sequence alignment of these CBS domains to translate these mutations
into the equivalent residues in CNNM3 CBS domains (Table 8).
49
50
We made nine point mutants in total and their locations are marked in the
sequence alignment of CNNMs CBS domains (Fig. 15).
Figure 15: Point mutations within the CNNM3 domains. We aligned the CBS domains of all CNNM family. As shown CBS domains are highly conserved and the point mutations we selected is conserve din all members.
We first performed the site mutagenesis in the CNNM3 2XCBS domain
instead of in the full length CNNM3 because we know that CBS domains
retain their structure and binding properties when separated from their
bulk protein (38). Seven point mutants were generated with Flag tagged
2xCBS constructs including Y301F, S334G, K360Q, D396A, E424A,
G433D and I445R (Table 8). Flag tagged CBS mutants were
co-transfected with GST-PRL-2 into HEK293 cells and IP with Flag (Fig.
16). TCL show expression of each CBS point mutant and GST PRL-2.
51
Since D396A and G433D mutations disrupted the interaction of PRL-2
with CNNM3 CBS domains. Subsequently, we generated CNNM3 full
length D396A, and G433D mutants to warrant the deleterious effect of
these mutations. We also included a CNNM3 full length T437N and
D439A mutant because of the conserved pathogenic role of these
residues in different CBS containing proteins (Table 8). Two pull down
assays were used. First, Flag bead was used to IP Flag tagged CNNM3
full length point mutants and GST PRL-2 (Fig. 17a). Consistent with
previous experiments, G433D disrupts CNNM3 interactions with PRL-2.
Surprisingly D396A mutants reduce the level of interaction but did not
abolish it. We repeated the experiment using a GST PD assay to verify if
this discrepancy results from an IP artifact. GST pull down was performed
on cells transfected with GST PRL-2 and Myc tagged CNNM3 full length
point mutants (Figure 17b). Similar results were obtained, confirming that
the G433D mutation completely abolished the interaction with PRL-2 while
D396A dramatically reduced the interaction. All together, these results
identified two key residues located in CNNM3 CBS domains that are
important for PRL-2 interaction.
52
Global phosphorylation did not promote CNNM3 interaction with PRL-2CSDA trapping mutant
The fact that PRL-2CSDA trapping mutant loses interaction with CNNM3
was surprising. Since a substrate must be phosphorylated in order to bind
to the catalytic pocket of the PTP, we want to know if the phosphorylation
of CNNM3 might promote its interaction with the trapping mutant. Thus,
we increased the global phosphorylation of all proteins by increasing the
activity of upstream kinase. We co-transfected the Hela cell with three
constructs, Flag CNNM3, GST PRL-2 or GST PRL-2CSDA and a
constitutively activated Src mutant. Although Src kinase increased overall
expression of Flag CNNM3, it did not affect its binding to PRL-2 and was
not trapped by PRL-2CSDA (Fig.18).
53
PRLs mutations disrupting their interaction with CNNM3
To investigate the reason why the PRL-2 C101SD49A trapping mutant
failed to interact with CNNM3, we tested the following PRL-2 mutants.
PRL-2 C101S (CS), D69A (DA), C101SD49A (CSDA), A108SC101S
(ASCS) and V110TC101S (VTCS) are mutants that inactivate PRL-2
phosphatase activity; A108S (AS) and V110T (VT) are mutants that
increase PRL-2 in vitro activity; the PRL-2 mutant with all tyrosine
phosphorylation sites mutated (6YF) has no known function. To ensure
disruption of these mutants was not the artifact of procedure, we
performed the IP in two separate systems. In the first method, GST pull
down was performed on cells transfected with GST-PRL-2 mutants and
Flag CNNM3 (Fig.19A). In the second method, Flag IP was performed on
a Flag PRL-2 mutant and Myc CNNM3 (Fig.19B). The results are
consistent in both assays, and show that CS alone completely eliminates
the interaction while the DA mutation impaired the binding greatly. In
addition, AS and VT mutations show the same binding as the wildtype
54
PRL-2. However, with addition of CS mutations these interactions were
completely abolished. These results demonstrate the important role of the
PRL-2 C101 and D69 residues play in for the interaction with CNNM3.
PRL-2 6YF mutants also demonstrated a dramatic decrease in interaction
compared to wildtype, suggesting that tyrosine phosphorylation of PRL-2
might regulate its interaction with CNNM3. Looking at other PRLs, we
performed a Flag IP assay on PRL-1 and PRL-3 mutants with equivalent
mutations. PRL-3 results are identical to PRL-2: CS and CSDA mutations
abrogate all interactions, while DA weakens the interaction. Unexpectedly,
PRL-1 CS, and DA mutants display almost equal binding as wildtype while
CSDA abruptly ends all interaction. C101 and D69 residues are important
for the PRLs-CNNM3 interaction and the structural difference between the
PRL-1 and PRL-3 active sites might account for the difference of CS and
DA mutations on interaction (39).
55
PRL-2 knockout mouse model
The oncogenic role of PRLs has been extensively researched; however
their exact biological functions remain unknown. In order to characterize
the physiological function of PRL-2, we generated a PRL-2 knockout
(KO) mouse model using PRL-2 gene trapped embryonic stem (ES)
cells purchased from the Sanger Institute. In PRL-2 gene trapped,
several elements are inserted into intron1 of PRL-2, including a splice
acceptor site (SA), the betaGeo gene and a transcription termination
sequence PolyA (Fig.20). With the insertion, the PRL-2 KO mouse
produces a truncated PRL-2 protein fused with betaGeo. A multiplex
PCR was performed as described in order to identify the different PRL-2
genotypes (Fig.20).
A western blot was performed on the genotyped mouse to confirm the
knockdown of PRL-2 at protein level (Fig.21). Results show a gradual
56
reduction of band intensity from wildtype mouse to knockout.
Interestingly, CNNM3 levels were not affected by the status of PRL-2.
We have previously tested the specificity of the PRL-2 antibody
(Fig.21B). The PRL-2 antibody is found to recognize both PRL-1 and
PRL-2, therefore the upper band in the PRL-2 blot belongs to PRL-1.
Immortalized MEF cell line
Murine embryonic fibroblasts (MEFs) are a great source of homogenous
stable cell lines that can be used in several cell based assays. MEFs
isolated from the PRL-2 knockout mouse provide a much more stable
and consistent PRL-2 knockdown compared to siRNA. Embryos from
heterozygote parents are harvested at E14.5 and trypsin digested to
isolate the primary MEF. Aliquots of cells were frozen and are currently
in the process of immortalization. We have successfully isolated
wildtype, heterozygote and knockout PRL-2 primary MEF lines as
57
shown by PCR genotype and western blot (Fig.22).
Reduced body weight of knockout PRL-2 mouse
We piloted a study on the body weight of the PRL mouse because we
observed KO mice are smaller in size compared to the wildtype mouse.
Several heterozygote crosses were designed to ensure a fair distribution
of wildtype, heterozygote and knockout in the offspring for comparison.
At 4 weeks old, we noticed a gradual decrease of the body weight from
WT to HET to KO (Fig.23). Moreover, the body weights of knockout
PRL-2 mice are significantly lower than their wildtype counterpart. This
result suggests a possible role of PRL-2 in metabolic signaling.
58
Chapter 4: Discussion
The PRL family belongs to the DSPs family of phosphatases, whose
expression is elevated in both metastatic cancer and primary tumors
(46). PRLs promote cell proliferation, migration and invasion. These
oncogenic properties have made PRLs a highly attractive target for
novel anticancer therapeutics (64). Although the involvement of PRL-2
in cancer has been investigated, little is known about its precise
physiological function, regulation, substrates and downstream signal
transduction pathways (93). Identification of PRLs substrates will be the
first step towards answering some of these questions, as well as to the
development of useful clinical specific inhibitors for PRL-2.
Identification of PRL-2 interacting partners using AP-MS
AP-MS is a newly developed technique used to identify protein
interacting partners. AP-MS coupled the mass spectrometry technique
directly after the Flag IP. The elimination of gel purification step allows
AP-MS to detect less abundant proteins, reduces sample loss from gel
extraction and avoids bias introduced from selecting stain bands for
analysis (17). Tables 4 and 5 contain all the potential candidates
identified in the AP-MS experiments; however the biological relevance of
these binding partners should be examined carefully for several
reasons. First, a previously reported PRL-2 binding partner RabGGTII is
not present in the results. The discrepancy can be accounted for by the
fact that RabGGTII’s interaction had only been found in an
59
overexpressed system (79). Second, in comparison to gel extraction
techniques, direct LC MS/MS analysis generates more non-specific
contaminants.The higher background can potentially mask the identity of
low abundance proteins (17). Third, although mass spectrometry can
determine the presence/absence of a peptide, it cannot detect the
relative difference in peptide binding. Several PRLs interacting partners
(i.e. actin and keratin) were identified in the PRL-2 IP results, however,
they were excluded as potential candidates because they are also found
in the control Flag empty IP (Table 3).
A literature search revealed an interesting relationships between several
of these proteins, including PRL-1, CKII, 14-3-3, TLS and YBX1. PRL-1
trimerizes in vivo and trimerization is essential for its ability to increase
proliferation and migration (84). The sequence and structure
configuration required for trimerization in PRL-1 is conserved in PRL-2
and -3, suggesting that trimerization is a shared regulatory mechanism
for all PRLs (84). Our AP-MS results showed an interaction of PRL-2
and PRL-2CSDA with PRL-1. We propose that PRL-1,-2, and -3 form
hetero-oligomers in vivo, and that the oligomerization of different PRL
members may dictate diverse regulatory mechanisms or substrate
specificities. CKII is a serine/threonine kinase, and the phosphorylation
of PTEN by CKII lead’s to its proteasome-mediated degradation. All
PRLs contain consensus CKII phosphorylation sites, suggesting that
CKII can regulate PRL-2 by phosphorylation (2). PRL-2 interacts with
three (ε, ζ/σ) out of seven isoforms of 14-3-3. 14-3-3 binds to
60
phosphorylated serine/threonine motifs on their target proteins (55). In
CDC25c, 14-3-3 binds and silences its NLS resulting in its cytoplasmic
retention (60). PRLs contain a polybasic region that functions as an
NLS, therefore, a 14-3-3 interaction might regulate the nuclear
localization of PRL-2 (84). YBX1 might be a downstream effector of
PRL-2. Similar to PRL-2, it regulates cell cycle progression, undergoes
nuclear localization and has elevated expression in breast and prostate
cancer (85). Interestingly, YBX1 also interacts with another PRL-2
interacting partner called TLS (also called FUS). YBX1 recruits the TLS
protein to pre-mRNA spliceosome machinery. Identification of both
proteins as PRL-2 interacting partners suggests PRL-2 is involved in the
RNA spliceosome complex (75). Pre-mRNA splicing is an essential
post-transcriptional RNA modification catalyzed by a large protein
complex spliceosome (12). snRNPs are important components of the
spliceosome; its biogenesis is mediated by the SMN complex containing
SMN, six Gemin proteins (Gemin2-7), and unrips (STRAP) (14)(15).
PRL-2 can be a component of the SMN complex, as PRL-2 was found
to interact with several components of this SMN complex, including
SMN, Gemin (3&6), and STRAP in our AP-MS results. In support of this,
previously published large scale mass spectrometry data also identified
STRAP and Gemin (3&4) as PRL-3 interacting partners (25) (Table 6).
SMN and Gemin3 subcellular localization is regulated by
dephosphorylation, suggesting that PRL-2 can be involved in the
regulation of the SMN complex (66).
61
CNNM3 interaction with PRL-2 and the PRL family
PRL-2 interacts with all members of the CNNM family (Table 7). We
validated the interaction of PRL-2 with one of its members, CNNM3, in
both endogenous and overexpression systems. CNNM3 was also found
to interact with all PRLs, suggesting that this interaction can be an
important regulatory mechanism conserved for all PRLs. CNNM3
belongs to a family of ancient conserved domain proteins (ACDPs or
CNNMs). CNNMs are closely located to one another on chromosomes 2
and 10, they are also similar in length and size (Fig.24).
CNNMs are expressed ubiquitously in all tissues. The highest
expression for CNNM2 is in the brain, kidney and placenta, in the heart
and spleen for CNNM3, and in the heart for CNNM4 (91). Their
ubiquitous expression patterns, conservation in divergent species, and
presence of multiple members in one species warrants the functional
importance of the CNNM family (90). CNNM members are involved in
divalent metal transport. Their homologue in bacteria, Corc, is
implicated in Mg2+ and Co2+ homeostasis. In yeast, Amip3 is implicated
in resistance to copper toxicity and, in S. cerevisiae, MAM3 is a
62
manganese resistant factor (80)(90)(98). CNMM members often
transport more than one type of metal; however, they all share their
greatest affinity for magnesium transport (5)(30)(80)(83)(98). CNNMs
physiological function was studied in different animal models. A
morpholino based expression knockdown of CNNM4 in zebrafish
showed defects in both heart and retinal ganglion cell development (72).
Also, mice placed on a low magnesium diet showed an up-regulation of
CNNM2 expression (30). The CNNMs family is clinically associated with
several diseases. All CNNMs were found by a genome-wide association
study to be involved in the regulation of magnesium homeostasis in
humans (53). CNNM1 is linked to urofacial syndrome (UFS) while
CNNM2 mutations result in dominant hypomagnesimia in humans
(5)(83). Lastly, mutations in CNNM4 cause Jalili syndrome, an
autosomal recessive disease of cone rod dystrophy (CRD) and
amelogenesis imeprfecta (AI) (32). The CNNMs family also has been
reported to be linked to cancer, CNNM1 is found to be hypermethylated
in melanoma cell lines (28). Although CNNM3 has been proposed to
share similar functions with other members of the family, their function
has not been studied. Our results therefore present the first insight into
the molecular function of CNNM3.
PRL-2 binds to CNNM3 at its CBS domains
All CNNM proteins contain four transmembrane and ACD domains. The
ACD domain is highly conserved within ACDPs, with approximately 92%
63
amino acid similarity (Fig. 15). Its sequence is also well conserved
among species, from bacteria, yeast, C. elegans, Drosophila to
mammals (91). The ACD domain contains two cysthathionine-beta-
synthase (CBS) domains, a cyclin box and a cylic nucleotide-
monophosphate-binding domain (cNMP binding domain) (Fig.12). The
presence of the cyclin box motif is the reason ACDP was first thought to
belong to a family of cyclin proteins (91). Cyclin proteins use this 31
conserved sequence motif to interact with CDKs (Ser/Thr protein
kinase) (61). The cNMP binding domain containing proteins includes
cGMP/cAMP dependent protein kinase (PKG/PKA), and cyclic
nucleotide gated channels (CNG). We have shown that PRL-2 interacts
with CNNM3 at its CBS domains. Since its discovery in 1997, more than
4,000 CBS domains have been found from archaebacteria to
eukaryotes (8). CBS domains are found in a wide range of proteins, and
they carry out very different roles (Table 9). CBS domains are regulatory
domains and sensors of cellular energy status. They also play a role in
oligomerization and subcellular trafficking (36). CBS domains act as
regulatory subunits in AMPK. The binding of AMP to the CBS domain
releases autoinhibitory regions in the kinase domain, allowing AMPK to
bind and phosphorylate its substrate (76). In many cases, CBSs are
thought to be sensors of cellular energy status, binding AMP/ATP/ADP
(76). Binding of an adenine nucleotide to the CBS domain activates both
IMDPH and mtCBS PPase (36)(38). Interestingly, CBS domains are
important for subcellular localization of chloride channels (CLC).
64
Truncation of CLC-5 CBS domains results in its retention in perinuclear
compartments instead of the acidic endosomes (36).
According to bioinformatics analysis, CBS dimers can interact with other
proteins. CLC5 containing a ‘PY’ motif between CBS dimmers
potentially interact with WW-domains of HECT-ubiquitin ligase (24). The
region between the CBS domains of IMPDH was proposed to bind
regulatory proteins (24). PRLs are the first protein identified to bind the
CBS domains.
Mutations in CBS domains result in several hereditary diseases,
including homocytstinuria (CBS protein), retinitis pigmentosa (IMPDH),
congenital myotonia (CLC-5), Wolf-Parkinson White syndrome (AMPKy)
and many more (Table 9). Interestingly, a single point mutation in
CNNM3 CBS domains completely abolished its binding to PRL-2
(Fig.17). G433D is a conserved residue in chloride channels (CLC). The
G433D mutation impedes CLC-2 ATP binding in vitro (76). Missense
mutations of this residue in CLC-1 results in muscular myotonia and
Albers-Schonberg disease in CLC-7, suggesting that this residue in
CLC CBS domains is critical for CLC function (73)(76). D396A is
another point mutation that dramatically affects the binding of PRL-2 to
CNNM3 CBS domains. D396A is a highly conserved residue within the
MgET CBS domain, a magnesium transporter closely related to the
CNNM family (34).
65
66
Several crystal CBS crystal structures are available, which includes
IMPDH (35), MgET(37), OpuA(51), CBS protein(54) and CLC (52).
Although CBS domain alignment shared very low sequence identity,
they shared common structural properties and conserved tertiary
structures (32). Each CBS domain contains 3 β-sheets and 2 α-helices
(76). Two CBS domains interact with their beta strand to form a globular
structure with a deep hydrophobic cleft (76). Alternatively, the two
amphipathic alpha helices contribute to the binding surface within this
hydrophobic cleft (24). We modeled the CNNM3 CBS domain with the
published structure of CLC-5 CBS domains using Multiple Sequence
Alignment by CLUSTALW (52) (Fig.25). The alignment sequence shows
conservation of some amino acid sequences, in particular in the second
and third beta strand. The CNNM3 G433 mutation is located in the third
beta strand and it is conserved in all of the CBS domains compared
(Fig.25).
67
68
PRL-2 catalytic domain mutations disrupts CNNM3 interaction
In the hope of better identifying PRL-2 substrates, we used the substrate
trapping mutant PRL-2CSDA in the AP-MS. The PRL-2 C101S mutation
allows binding of a substrate but blocks the catalysis, such that the
substrate is not released, thus enhancing the enzyme substrate
interaction (9). The D96A mutation stabilizes the WPD loop that comes
over the catalytic pocket to prevent the release of the substrate (9).
Considering the function of these mutants, if CNNM3 is a true substrate, a
stronger interaction should be detected in trapping mutants compared to
wildtype as illustrated in the interaction of PRL-3CS with NCL and KRT8
(57)(78). Alternatively, if CNNM3 is an interacting partner, binding of
CNNM3 to PRL-2CSDA should remain at the same level as wild type
PRL-2. Surprisingly, not only did the CSDA mutation not enhance the
CNNM3 interaction with PRL-2, it abolished the interaction completely.
The cysteine and aspartic acid are the two most important residues
required for PRL-2 catalysis, they are conserved in all PTPase and are
critical for phosphatase activity (9). C101 acts as a nucleophile to attack
the phosphorus centre of the substrate, and D96 acts as a general acid to
protonate the release of substrate (9). Additional PRL mutations showed
that the CS mutation alone completely abolished PRL-2 interaction and
that the DA mutation decreased the strength of the interaction. On the
other hand, AS and VT mutations did not affect CNNM3 binding at all.
PRL-2 A108 and V110 are two residues located close to the catalytic
motif, but they are not critical for PRLs catalytic function (Fig. 26). In fact,
69
the presence of the A108 and V110 in place of S108 and T110
respectively is the reason why PRLs have much lower catalytic activity
compared to other PTPs (84). Our results suggest specific interactions of
CNNM3 to PRL-2 D96 and C101 residues, the two residues that are
critical for PRL phosphatase activity. Additional mutations within the WPD
and PTP catalytic motif will be used to confirm the specificity of this
interaction.
Model for CNNM3 PRL-2 interaction
The observation that CNNM3 binds only to wildtype PRL-2, but not the
trapping mutants, suggests that CNNM3 is not a substrate but a PRL-2
interacting partner. The lack of a regulatory domain in PRLs and their
remarkably low intrinsic phosphatase activity suggests that the PRLs are
regulated by their interacting proteins. Interacting proteins can act as
regulatory subunits to enhance PRLs phosphatase activity (22). Also,
PRLs activity may not depend on its phosphatase activity at all but rather
on its interaction with other proteins (59). The fact that CNNM3 is not a
substrate but binds to the catalytic domain of PRL-2 suggests its role as a
regulatory protein inhibiting PRL-2 phosphatase activity. I propose that
70
CNNM3 CBS domains (G433) bind to the catalytic pocket of PRL-2 via
C101 and D69, preventing the binding of PRL-2 substrates, therefore
inhibiting PRL-2 phosphatase activity. To validate our model, we need to
confirm the direct interaction of PRL-2 with CNNM3 at its catalytic domain.
Several experiments can be carried out. First, direct interaction of PRL-2
and CNNM3 can be demonstrated in an in vitro binding system consisting
only of bacteria purified PRL-2 and CNNM3. Second, phosphatase activity
assays can measure the dosage dependent inhibition of PRL-2
dephosphorylation activity by CNNM3 binding or just CBS binding. Third,
we want to generate a crystal structure of the CBS domain in complex
with PRL-2. Finally, we will perform immunofluorescence studies to
confirm the co-localization of these proteins.
Although we have shown that PRL-2 only interacts with CBS domains of
CNNM3, several studies suggest that other domains of CNNM3 might play
role in regulating this interaction. First, CNNM4 missense mutations
outside the CBS domain are implicated in Jalili syndrome, suggesting that
other domains are essential for CNNM4 function (63). Second, the CBS
domain alone lacks substrate specificity. CBS domains of CLC channels
are able to functionally substitute one another as well as with the CBS
domain of IMPH2, suggesting that other domains might contribute to the
regulatory and/or substrate recognition function of the CBS domain (24).
The cyclic nucleotide binding domain is another interesting domain found
in CNNM3. cNMP binding domains in cyclic nucleotide-gated (CNG) ion
channels are modulated by cellular cAMP levels. Binding of cAMP to a
71
cNMP binding site changes the conformation of the CBS domain,
activating the ion channels (77). It will be interesting to test if the addition
of cNMP in vitro will affect CNNM3 and PRL-2 interactions.
Physiological role of PRL-2
In this study, we successfully generated a knockout mouse model of PRL-
2. A significant reduction in body weight was detected in the PRL-2
knockout mouse compared to its wildtype counterpart, suggesting that
PRL-2 might play an important physiological role. Further analysis of the
PRL-2 KO mouse model will reveal the exact cause of this weight loss.
This model can also be used to study the mechanism of action of the PRL-
2 and CNNM3 interaction. Knockdown of PRL-2 in thymic tissues and
MEF did not affect the level of CNNM3, suggesting that PRL-2 does not
regulate CNNM3 protein levels. We will perform western blot analysis to
investigate the effect of PRL-2 knockdown on CNNM3 level in other tissue
samples. CNNM3 and PRL-2 interaction may be involved in the
phototransduction cascade in cone photoreceptors. CNNM4 mutations
cause cone-rod dystrophy (CRD), a disease associated with loss of cone
compared to rod photoreceptors (63). On the other hand, PRL-1 is found
to be a molecular component of the cone photoreceptor’s response to
oxidative stress. Its expression is preferentially localized to the cone
photoreceptor cell outer segment (100)(102). PRLs are normally
associated with the cytoplasmic face of plasma membranes therefore they
may function to modulate membrane channels or participate in the
72
regulation of the phototransduction cascade (100). I anticipate that PRL-2
interacts with CNNM3, a metal transporter in the plasma membrane of
cone photoreceptors and that this interaction modulates the
phototransduction cascade. As mentioned above, the cNMP binding
domains are found in CNNM3 and CNG. CNGs are photoreceptors found
in the outer segment of rod photoreceptors, they are essential for the
generation of primary electrical signals in response to light. Binding of
cAMP to cNMP binding sites change the conformation of the CBS domain,
mediates the influx of calcium and sodium (77). Thus, we hypothesize that
cNMP might also regulate the interaction of PRL-2 and CNNM3 in cone
photoreceptors. Light elevates the cNMP level in the cell, increasing the
binding of cGMP which changes the conformation of the CBS domain,
releasing it from the catalytic domain of PRL-2 and activating its
phosphatase activity downstream (63). The first step to validate our
hypothesis will be investigate our knockout mouse models to see if there is
retinal degeneration, in particular any abnormality or loss of cone
photoreceptors. Second, we want to perform immunostaining of both PRL-
2 and CNNM3 to see if they co-localize to the same region in the
membrane of cone receptors. In addition, the CNNMs family also plays a
role in magnesium transport. CNNM2 mutations are associated with
dominant hypomangesmia, an abnormal urinary Mg2+ excretion. It would
will be interesting to measure the urinary content of Mg2+ in the PRL-2 KO
mouse. Mice kept at low magnesium diet have shown up-regulation of the
CNNM2 transcript, therefore we can also challenge our mouse with a low
73
magnesium diet (94).
Conclusion
We have identified several putative candidates proteins that with PRL-2
using AP-MS. Furthermore, the interaction of PRL-2 with one of these
candidates: CNNM3 was validated using co-IP. We identified CNNM3 as
the first interacting partner shared by all PRL members and PRLs as the
first proteins that interacts with CBS domains. We characterized and
proposed a model for the function of the CNNM3 and PRL-2 interaction.
We also suggested their possible physiological function in cone
photoreceptors. In addition, we generated a PRL-2 knockout mouse
model and found significant reduction in their body weight, suggesting an
important physiological role of PRL-2. The identification of PRL-2 partners
will lead to a better understanding of its cellular mechanisms and the PRL-
2 KO mouse model will provide us with an excellent tool to study its
physiological function.
74
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