Cdc2-mediated Phosphorylation oftheGapJunction Protein ...

Vol. 9, 13-21, January 1998 Cell Growth & Differentiation 13

Cdc2-mediated Phosphorylation of the Gap Junction Protein,Connexin43, during Mitosis1

Martha Y. Kanemitsu,2 Wei Jiang, andWafter EckhartMolecular Biology and Virology Laboratory, The Salk Institute for

Biological Studies, San Diego, California 92186

AbstractAs cells enter mitosis, gap junctional communicationwith neighboring cells decreases (H. Xie et aL, J. CellBiol., 137: 203-210, 1997). Phosphorylation of the gapjunction protein, connexin43 (Cx43), has been

implicated in reducing junctional permeability. Cx43contains ,�cdc2 phosphorylation consensus sites in itsCOOH-terminal region. Accordingly, we examined therole of p34CdC2/cyclin B in Cx43 phosphorylation.Purified p34�/cyclin B, or p34cd02/cyclin B compleximmunoprecipitated from mitotic cells, phosphorylatedGSTCx43 in vitm. The synthetic peptide,SDPYHATrGPLSPSKDCGSPK, corresponding to aminoacids 241-264 of Cx43, was also phosphorylated byp34�/cycIin B in vitro. Sites phosphorylated in vitrowere phosphorylated in vivo. Butyrolactone I, aninhibitor of cdc2 kinase, inhibited increases in Cx43phosphorylation during mitosis. We conclude thatphosphorylation of Cx43 by p34#{176}�/cyclin B maycontribute to the increased Cx43 phosphorylation andreduced gap junctional communication observedduring mitosis.

IntroductionAs cell enter mitosis, dramatic changes in cell structureoccur, including nuclear envelope breakdown, chromosomal

condensation, formation of the mitotic apparatus, and re-organization of the cytoskeletal architecture (reviewed inRef. 1). Recently, it was demonstrated that gjc3 was reducedduring mitosis (2, 3).

Intercellular communication, mediated by gap junctionchannels, coordinates cellular processes including contrac-

Received 6/27/97; revised I 0/9/97; accepted I 1/3/97.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 This work was supported by USPHS Grants CA09370, CA13884, andCA14195 from the National Cancer Institute, NIH, and Grants ACS-PF-4275 (to M. Y. K.) and ACS-PF-4274 (to W. J.) from the American CancerSociety.2 To whom requests for reprints should be addressed, at Molecular Biol-ogy and Virology Laboratory, The Salk Institute, P. 0. Box 85800, SanDiego, CA 92186-5800. Phone: (619) 453-4100; Fax: (619) 457-4765;E-mail: [email protected] The abbreviations used are: gjc, gap junctional communication; cdk,cyclin-dependent kinase; Cx43, connexin 43; GST, glutathione S-trans-ferase; MAP, mitogen-activated protein; CK, casein kinase; IP, immuno-precipitation; PAP, potato acid phosphatase.

tion of cardiac and smooth muscle (4, 5), synaptic transmis-sion of electrical impulses (6), embryogenesis (7, 8), andgrowth control (reviewed in Refs. 9 and 1 0). Gap junction

channels, which connect the cytoplasms of adjacent cells,allow the passage of ions, second messengers, and smallmolecules less than Mr 1000 (1 1).

Gap junctions are formed by specialized proteins called

connexins. Six connexin molecules assemble in the lipidbilayer of the plasma membrane to form a connexonhemichannel. Each connexin protein has four membrane-spanning regions, a cytoplasmic loop, two extracellular

loops, and cytoplasmic NH2- and COOH-terminal ends.Hemichannels from adjacent cells join to form a continuousaqueous pore (11).

Connexins are encoded by a multigene family. Thirteenfamily members have been identified (1 1). Some connexinsare widely expressed, whereas others exhibit restricted cell

type and tissue distribution, suggesting that there are func-tional differences among connexin family members (1 1). TheCOOH-terminal region is highly variable among connexins.Some connexins have consensus protein kinase phospho-rylation sites in their COOH-terminal regions (12-14). InCx43, multiple protein kinase phosphorylation sites havebeen identified (1 3). Phosphorylation of the protein has beenimplicated as a regulatory mechanism for channel gating(reviewed in Refs. 13 and 15). Reduced gjc during mitosis isaccompanied by a rapid increase in Cx43 phosphorylation(3).

Protein phosphorylation, which is important for cell cycle

progression, is controlled, in part, by a family of proteinkinases, the cdks (1). Among the cdks, p34cd02 plays animportant role in the initiation of mitosis (1). Several proteins

involved in mitosis-specific cellular changes become newlyphosphorylated or hyperphosphorylated during mitosis, in-cluding histone Hl , nucleolin, nucleoporin, plectin, myosin-Il

regulatory light chain, vimentin, and nuclear lamins (reviewedin Refs. 1 6-1 8). These proteins are in vivo and/or in vitro

substrates of p34cdc2 kinase (reviewed in Refs. 16-18).Comparison of the amino acid sequences of substrates of

p34cdc2 have identified S/TPXK/R or SPK as consensusphosphorylation sequences (1 6, 18). Sequence analysis ofCx43 shows p34cdc2 consensus phosphorylation sites atresidues 252-255 and 262-264. Therefore, hyperphospho-rylation of Cx43 during mitosis may be mediated by p34�2.

In the present study, we examined Cx43 phosphorylation

by p34cdc2#{149} Cx43 phosphorylation increased dramaticallyduring mitosis, predominantly on senne residues. p34c�

kinase activity also increased during mitosis. In addition,purified or immunoprecipitated p34c&2/cyclin B complexphosphorylated a GST Cx43 fusion protein and a syntheticpeptide containing both COOH-terminal p34c� consensusphosphorylation sites in vitro. Butyrolactone I, an inhibitor ofp34cdc2 kinase activity, blocked the ability of p34cdc2/cyclin

/‘p � c;.. c’, (4�

*-HC IgG

1 2345 6789

A.

B.

C. PAA: � I I I IO�6 : � � ‘�. .,O�-� � � :� Ct

I I � I ID. FACS AnalysIs:

G1(%) 62 91 80 11 4

S(%) 19 4 12 85 6G2IM(%) 19 5 8 4 90

Fig. 1 . Cx43 phosphorylation increases dur-ing mitosis. A, lysates of Rat-i fibroblasts,either asynchronously growing (Asyn) or syn-chronized at various stages of the cell cycle(G0, G1-S, 5, G2-M, G2, and M phases), werenormalized for protein content and precipi-tated with 1 �.tg of aCx43 antiserum. (Noc),cells were treated with nocodazole (1 00 ng/ml). Cx43 immunoprecipitates from mitoticRat-i fibroblasts were dephosphorylated byincubation with PAP [M(Noc)+P’asej. The im-munoprecipitated proteins were separated bySDS-PAGE, electrotransferred to ImmobilonP membrane, and immunoblotted with aCx43antiserum. The nonphosphorylated (NP� andphosphorylated (P1, P2, and P�) forms ofCx43 are as indicated. Arrow, heavy chain ofimmunoglobulin (HC lgG). B, in parallel, Rat-lfibroblasts were metabolically labeled with[�2P]P,, solubilized in RIPA buffer, and immu-noprecipitated with aCx43 antiserum. The im-munoprecipitates were separated by SDS-PAGE and autoradiographed. 32P-labeledCx43 proteins were isolated, and 32P incor-poration was quantified by Cerenkov count-ing. The values are expressed relative to Cx43phosphorylation during G1-S phase. Eachpoint represents the average of two inde-pendent experiments; bars, SE. C, phospho-rylated Cx43 proteins were further analyzedto determine phosphoamino acid content.The positions of phosphoserine (P-S), phos-phothreonine (P-i), and phosphotyrosine(P-Y) are indicated. D, asynchronously grow-ing and synchronized Rat-i fibroblasts wereverified for cell cycle position by propidiumiodide staining and flow cytometry. The num-hers shown represent the percentage of thecell population (5 x 1O� cells).

14 Cx43 Phosphorylation in Mitotic Cells

p3

P1-p

B complex to phosphorylate Cx43 in vitro. The inhibitor also

blocked in vivo increases in Cx43 phosphorylation during

mitosis. Two-dimensional tryptic phosphopeptide mapping

demonstrated that phosphopeptides generated from the

synthetic Cx43 peptide after phosphorylation in vitro by pu-

rified p34cdc2/cyclin B complex were a subset of phos-

phopeptides generated from Cx43 phosphorylated in vivo

during mitosis. We conclude that p34�2 mediates mitotic-

specific phosphorylation of Cx43 on serine 255 and possibly

serine 262.

ResultsPhosphorylation of Cx43 Increases during Mitosis. To

examine the phosphorylation of Cx43 at different stages of

the cell cycle, Cx43 was immunoprecipitated from cellular

lysates of asynchronous or synchronized Rat-i fibroblasts

and analyzed by aCx43 immunoblotting. As shown in Fig.

1A, multiple species of Cx43 proteins were observed from

asynchronous Rat-i cells (Lane 1) or cells synchronized atthe various stages of the cell cycle (Lanes 2-9). The fastest

migrating species of Cx43 is the nonphosphorylated form,

whereas the slower migrating species (P1 , P2, and P3) arephosphorylated forms (1 9-21). The slowest migrating, phos-

phorylated forms of Cx43 (Ps) were more abundant in no-

codazole-treated cells blocked at the G2-M transition of the

cell cycle (Fig. 1A, Lane 5). Cx43 phosphorylation during

G2-M transition was further analyzed by separating cells in

G2 and M phases of the cell cycle. The slowest migratingform of Cx43 was more abundant in M phase than in G2

phase (Fig. 1A, compare Lanes 6 and 7). Treatment of Cx43

from M phase cells with potato acid phosphatase resulted in

loss of the slowest migrating form and accumulation of thefastest migrating form (Fig. 1A, Lane 8), suggesting that the

migration shift observed during M phase was due to phos-

C,Co � � (1�

l�. (, (, C, 0W

4 cdc2/cyclin B complex was purified from Sf9 cells coinfected with cyclinB and cdc2 baculoviruses. W. Jiang, G. Jiminez, N. Wells, G. WahI, T.Hunter, and R. Fukunaga. A mitotic-spindle-associated protein requiredfor cytokinesis, manuscript in preparation.

Cell Growth & Differentiation 15

acdc2 lP-ln vitro kinase

“�-Histone Hi

‘#{248}-GSTCx43 Tail

Fig. 2. Mitosis-specific phosphorylation of Cx43 by p34cdc2 j� vitro.cdc2/cyclmn B complex was immunoprecipitated from lysates of asyn-chronously growing (Asyn.) or synchronized (G0, G1-S, S, and G2-Mphases) Rat-i fibroblasts and used for in vitro kinase reactions withhistone Hi and GST Cx43 COOH-terminal tail fusion proteins as sub-strates. The kinase reactions were terminated by the addition of 2xsample buffer, separated by SDS-PAGE, and autoradiographed. Arrows,phosphorylated histone Hi and GST Cx43 tail.

phorylation of the protein. The slowest migrating form of

Cx43 was also observed in mitotic cells in the absence ofnocodazole, indicating that the mitotic-specific increase in

Cx43 phosphorylation was not the result of nocodazole treat-ment (Fig. iA, Lane 9).

To examine levels of Cx43 phosphorylation and phos-phoamino acid content, asynchronously growing or synchro-

nized Rat-i fibroblasts were metabolically labeled with

[32P]P. 32P-labeled Cx43 was immunoprecipitated from celllysates, resolved by SDS-PAGE, and quantified by Cerenkovcounting. Cx43 phosphorylation increased 4.5- and 2.5-fold

during G2-M and S phases, respectively, relative to the level

of phosphorylation observed during G1-S phase (Fig. 1B). Nodifferences in Cx43 phosphorylation were observed between

G0 and G1-S-phase cells (Fig. iB). The 32P-Iabeled Cx43

proteins were analyzed for phosphoamino acid content.

Cx43 was phosphorylated predominantly on phosphoserinethroughout the cell cycle. Trace amounts of phosphothreo-

nine appeared in G2-M phase (Fig. 1 C). The cell cycle posi-tion of Rat-i cells was verified by flow cytometry analysis

(Fig. 1D). Therefore, Cx43 phosphorylation occurs predom-inantly on serine residues and varies in a cell cycle-depen-

dent manner.

In Vitro Phosphorylation of Cx43 by p34cdc2 from Mi-totic Cells. The COOH-terminal tail of Cx43 contains con-sensus phosphorylation sequences for p34cdc2 at amino acid

residues 252-255 (SPSK) and 262-264 (SPK). Because

p34cdc2 is active during mitosis, we tested whether Cx43

could serve as a substrate of p34cdc2. p34cdc2/cyclin B com-

plexes immunoprecipitated from asynchronous or synchro-nized Rat-i fibroblasts were used for in vitro kinase reactions

with histone Hi and GST Cx43 COOH-terminal tail fusion

protein (residues 236-282) as substrates. As shown in Fig. 2,

phosphorylation of histone Hi by p34cdc2/cyclin B complex

in vitro was maximum during G2-M phase (top panel). Sim-

ilarly, phosphorylation of GST Cx43 tail was maximum during

G2-M phase (Fig. 2, bottom panel). These results indicatethat Cx43 can serve as a substrate for p34cdc2 in vitro, and

that phosphorylating activity is highest during G2-M phase.

p34cdc2/Cyclin B Complex Phosphorylates a PeptideCorresponding to Amino Acids 241-264 of Cx43. To fur-ther characterize Cx43 phosphorylation by p34cdc2, a syn-thetic peptide containing both p34cdc2 consensus phospho-

rylation sites (Fig. 3A, amino acids 24i-264) was used as a

substrate in in vitro kinase reactions with purified p34cdc2/

cyclin B complex.4 Phosphorylation of the synthetic Cx43

peptide was not observed when in vitro kinase reactions

contained either purified p34cdc2/cyclin B complex or pep-

tide alone (Fig. 3B, left panel). However, in the presence of

both components, the synthetic Cx43 peptide was highly

phosphorylated (Fig. 3B, left panel). Similarly, the synthetic

Cx43 peptide was highly phosphorylated by p34cdc2/cyclin Bcomplex immunoprecipitated from G2-M phase cells but not

from asynchronous, G0, G1 -5, or S-phase cells (Fig. 3B, right

panel).

Inhibition of Mitotic-specific Increases in Cx43 Phos-phorylatlon by Butyrolactone I, a p34�’�2 Inhibitor. Pre-vious studies demonstrated inhibition of in vitro and in vivo

p34cdc2 activity by butyrolactone I (22-24). Butyrolactone I

inhibited cdc2 and cdk2 kinases but had little effect on

mitogen-activated protein kinase, protein kinase C, casein

kinases I and II, protein kinase A, or epidermal growth factor

receptor tyrosine kinase (22). In addition, butyrolactone I was

selective among cdk family members. cdc2, cdk2, and cdk5

kinases displayed sensitivity toward butyrolactone I,

whereas cdk4 and cdk6 kinases were insensitive (reviewed in

Ref. 24). We first examined whether butyrolactone I could

have an effect on the banding profile of Cx43 by immunoblot

analysis. Because cdk2 activity is high at late G1 and early S

phase, butyrolactone I was added as cells entered G2 phase

to ensure that cdc2 but not cdk2 kinase activity was inhib-

ited. In the presence of butyrolactone I, the slower migrating,

highly phosphorylated form of Cx43 was absent, and the

faster migrating, nonphosphorylated form of Cx43 was pre-

dominant (Fig. 4A). As shown in Fig. 4B, fluorescence-acti-

vated cell sorting analysis demonstrated that Rat-i cells

pretreated with butyrolactone I prior to nocodazole treatment

were predominantly blocked at G2-M, similar to that ob-

served in the absence of butyrolactone I. However, the cells

did not round up and arrest in mitosis but remained flat and

adherent in the presence of butyrolactone I (data not shown),

suggesting that cdc2 activity was inhibited during G2-M, and

cell cycle progression into mitosis was blocked by butyro-

lactone I. To examine whether butyrolactone I could inhibit in

vitro phosphorylation of GST Cx43 tail by immunoprecipi-tated p34cdc2/Cyclin B complex, in vitro kinase reactions

were performed in the presence or absence of butyrolactoneI. As shown in Fig. 4C, phosphorylation of the GST Cx43 tailby p34cdc2/cyclin B complex was 1 2-fold higher by the com-

plex immunoprecipitated from mitotic cells than by the corn-


A.241 264

SDPYHATTG PL�PSKDCG�PK255 262

B.

cdc2/cyclln B:peptlde:

+ +

- + +

‘#{248}-peptide -*#{176}

Purified cdc2/cyclln B czcdc2 IP

Fig. 3. p34cdc2 kinase phosphorylates a synthetic peptide containing serine 255 and serine 262 of Cx43 in vitro. A, amino acid sequence ofthe syntheticpeptide corresponding to residues 241-264 of Cx43. p34cdc2 phosphorylatlon consensus sequences are shown in bold and putative p34C�2 phospho-rylation sites are underlined. In B, in vitro kinase reactions were performed in the presence of purified cdc2/cyclin B complex4 alone, synthetic peptide alone,or both components (left panel). p34cdc2 immunoprecipitated from lysates of asynchronously growing (Asyn.) or synchronized (G0, G1-S, 5, and G2-Mphases) Rat-i fibroblasts was incubated with the synthetic peptide (rlghtpanel). The kinase reactions were terminated by the addition of 2x sample buffer,separated on a 20% polyacrylamlde gel by SDS-PAGE, and autoradlographed. Arrows, the phosphorylated synthetic peptides.

plex from interphase cells. In the presence of butyrolactoneI, phosphorylation by the complex from mitotic cells was only

3-fold higher than by the complex from interphase cells

(Fig. 4C).To examine whether butyrolactone I affected the increase

in Cx43 phosphorylation during rnitosis, Rat-i fibroblasts

metabolically labeled with [32P]P� were treated with butyro-

lactone I (236 fLM) 5 h after release from G1 -S block (1 h prior

to the addition of nocodazole). 32P-Iabeled Cx43 proteins

were immunoprecipitated, resolved by SDS-PAGE, andquantified. As shown in Fig. 4D, phosphorylation of Cx43

increased 4-fold during mitosis relative to the basal phos-phorylation observed during interphase. Butyrolactone I in-hibited mitotic-specific increases in Cx43 phosphorylation

(Fig. 4D). Phosphorylated Cx43 proteins were isolated, and32p incorporation was quantified by Cerenkov counting;thedata (cpm) for two independent experiments are shown inFig. 4E. These results provide further evidence that p34cdc2

mediates the increase in phosphorylation of Cx43 duringmitosis.

Cx43 Sites Phosphorylated by p34C�2 jj Vitro Are aSubset of Sites Phosphorylated in Vivo during Mitosis.To examine whether Cx43 is phosphorylated at similar sites

in vivo during mitosis and in vitro by purified p34c�2/cycIin B

complex, two-dimensional tryptic phosphopeptide mappingwas performed. As shown in Fig. 5A, several phosphopep-

tides of Cx43 were observed from interphase Rat-i cells. In

contrast, the map of Cx43 from mitotic Rat-i cells revealed

dramatic increases in the number and intensity of phos-

phopeptides (Fig. SB). In particular, the intensities of phos-

phopeptides 1 , 2, and 3 were increased, and phosphopep-tide 4 appeared (Fig. SB). Three major phosphopeptides

were generated from the synthetic Cx43 peptide phospho-rylated in vitro by purified p34cdc2/cyclin B complex (Fig. SC).

The predicted mobility of the synthetic Cx43 tryptic phos-

phopeptides was determined and plotted based on theamino acid composition of the peptide, as described previ-

ously (25). It implicated phosphopeptide 2 as the fragment

containing serine 255, phosphopeptide 4 as the fragment

containing serine 262, and phosphopeptide 3 as the undi-

gested peptide containing both putative p34cdc2 phosphorylation sites (Fig. SD). The low radioactive content of in

vivo-Iabeled endogenous Cx43 combined with the high level

of radioactivity in the in vitro-phosphorylated synthetic pep-

tide made loading equal counts in the mixing experimentdifficult. Despite unequal loading, the mixing experiment

demonstrated that in vitro phosphopeptides 2 and 3 were asubset of phosphopeptides generated from Cx43 phospho-rylated in vivo during mitosis (Fig. SE). Phosphopeptide 4generated from the synthetic peptide phosphorylated in vitro

by purified cdc2/cyclin B complex did not comigrate withphosphopeptide 4, which appeared during mitosis but in-

stead comigrated with a minor phosphopeptide migrating

just above phosphopeptide 4 (Fig. 4E). The reason for thisdiscrepancy is unclear. Therefore, it is uncertain whetherphosphopeptide 4, which increased during mitosis, contains

serine 262. Closer examination by manual Edman degrada-

tion should clarify the identity of phosphopeptide 4. In the

presence of butyrolactone I, the intensities of Cx43 phos-phopeptides from mitotic Rat-i cells were diminished (Fig.SF). These results support the conclusion that p34cdc2 phos-phorylates Cx43 during mitosis on serine 255 and possiblyserine 262.

Discussiongjc decreases during mitosis (2, 3). The effect is transient,

and gjc is reestablished soon after cytokinesis (2). Reducedgjc during mitosis is accompanied by increased phosphoryl-

ation of Cx43 (3). The kinase(s) responsible for phosphor-

A. WI

pI ]Cx43

B.

D.C.

1 2

In vitro In vivo

#{149}xpt.1 1777 18937 3830expt. 2 8315 109887 36209

I M M+B

1275 5600 1205367 1350 467

Fig. 4. Inhibition of mitosis-specific increases in Cx43 phosphorylationby butyrolactone I, an inhibitor of p34�2. In A, Cx43 was immunopre-cipitated from nocodazole-treated (1 00 ng/m� Rat-i fibroblasts in theabsence (M) or presence (M + But.) of 236 �.ui butyrolactone I. Theimmunoprecipitated proteins were separated by SDS-PAGE, electro-transferred to Immobilon P membrane, and immunobiotted with aCx43antiserum. Arrow, heavy chain of immunoglobulin (HCIgG). Bracket, Cx43proteins. B, in parallel, cell cycle position of nocodazole-treated (100ng/mI) Rat-i cells in the absence (M) or presence (M + But.) of 236 �.tM

butyrolactone I were analyzed by flow cytometry. The numbers shownrepresent the percentage of the cell population (5 x iO� cells). In C, GSTCx43 COOH-terminal tail fusion protein was phosphorylated in vitro byp34cdc2 immunoprecipitated from Rat-i fibroblasts in interphase (I) ornocodazole-arrested in mitosis (M). Kinase reactions were conducted inthe absence (I and M) or presence (M + B) of 62 �ui butyrolactone I.32P-labeled Cx43 was immunoprecipitated from lysates of Rat-i fibro-blasts metabolically labeled during interphase (I) or while nocodazole-arrested in mitosis in the absence (M) or presence (M + B) of 236 �ui

butyrolactone I. Phosphorylated Cx43 proteins were resolved by SDS-PAGE and autoradiographed. 32P-labeled Cx43 proteins were isolated,and 32P-incorporation was determined by Cerenkov counting. The valuesare expressed relative to the 32P content of Cx43 from interphase cells.Each point represents the average of two independent experiments; bars,SE. E, in vitro and in vivo Cx43 phosphorylation (cpm) as determined byCerenkov counting.


c 15

I 10

H

5

II M M+B

E. I I I

G1(%) 3 12S(%) 6 17G2/M (%) 91 71

ylation of Cx43 during mitosis has not been identified

previously.

The results reported here demonstrate that p34cdc2 phos��phorylates Cx43 during mitosis in Rat-i fibroblasts. The ev-idence can be summarized as follows. Cx43 phosphorylation

and p34cdc2 kinase activity increased coordinately duringG2-M (Figs. i and 2). p34cdc2 from mitotic cells phosphoryl-

ated a GST Cx43 COOH-terminal tail fusion protein in vitro

(Fig. 2). Phosphorylation of the p34cdc2 consensus phospho-rylation sites in a synthetic peptide corresponding to resi-

dues 241-264 of Cx43 by purified p34cdc2 in vitro (Fig. 3B)

produced phosphopeptides 2, 3, and 4, identified by two-dimensional tryptic phosphopeptide mapping (Fig. SC).Phosphopeptides 2, 3, and 4 are a subset of in vivo phos-phopeptides (Fig. SE), suggesting that they are physiological

,�; targets of p34cdc2 Butyrolactone I, a specific inhibitor of

� p34cdc2, inhibited increases in Cx43 phosphorylation in vitro

and in vivo (Figs. 4 and SF).In addition to phosphopeptides 2, 3, and 4, phosphopep-

tide 1 was observed in tryptic phosphopeptide maps of Cx43

phosphorylated in vivo during mitosis. Phosphopeptide 1

also exhibited a dramatic increase in phosphorylation duringmitosis. Phosphopeptide 1 may be a partial digestion prod-uct containing the p34cdc2 phosphorylation sites. Altema-tively, phosphopeptide i may be a target of other serine/

threonine kinases. Cx43 has been identified as a substrate

for MAP kinase, protein kinase C, and cAMP-dependentprotein kinase (26-28). Cx43 also contains phosphorylation

consensus sequences for CKI and CKll and glycogen syn-thase kinase-3 (28-30). In addition to the direct effects of

these kinases on Cx43 phosphorylation, indirect effects arealso possible. For example, CKII is phosphorylated by

p34cdc2 during mitosis (3i), and MAP kinase inhibits the

inactivation of p34cdc2 kinase/cyclin B in G2-arrested

oocytes (32). Therefore, Cx43 may be a target of multiple

kinases during mitosis, resulting in hyperphosphorylationand subsequent disruption of gjc.

Some sites of phosphorylation in Cx43, in particular serine255, are candidates for phosphorylation by more than onekinase. Serine 255 in phosphopeptide 2, which is phospho-rylated by p34cdc2 is also phosphorylated by MAP kinase in

response to epidermal growth factor stimulation of TSi B ratliver epithelial cells (28, 33). Several proteins important for

cell cycle progression (Elk-i ; Sapia; lamins A, B, and C;

weei +; and myc), in addition to Cx43, contain overlappingMAP kinase and p34cdc2 kinase phosphorylation consensus

sequences (PXS/TPXK/R), suggesting that MAP kinase andp34cdc2 kinase could have common substrates (reviewed inRef. 16).

Another kinase that potentially could phosphorylate Cx43

during mitosis is cSrc. Cell cycle-dependent activation ofcSrc has been reported (34-36). cSrc activity increases dur-ing G0-G1 transition following growth factor stimulation (37-39) and during mitosis (34, 40). Cell division was blocked

when cells were microinjected during G2 with a neutralizing

antibody specific for Src family members Src, Fyn, and Yes,

suggesting that Src is required for entry into mitosis (40).

Tyrosine phosphorylation of Cx43 is increased, and gjc re-duced, in cells transformed by vSrc or expressing constitu-tively-activated cSrc (F527 cSrc; Refs. 1 9, 41 , and 42).Therefore, it was plausible that Src, activated during mitosis,might phosphorylate Cx43 on tyrosine and contribute to loss

of gjc. Consistent with previous reports (3), increased levels

of Cx43 phosphorylation were observed in mitotic Rat-ifibroblasts (Fig. 1 , A and B). However, phosphoamino acid

analysis of 32P-Iabeled Cx43 demonstrated that Cx43 is

phosphorylated predominantly on serine, with trace amounts

of phosphothreonine, and no detectable phosphotyrosine

found during mitosis (Fig. iC). Moreover, tyrosine phospho-

rylation of Cx43 during mitosis was not detectable bya-phosphotyrosine immunoblot analysis (data not shown).

Similar results (lack of tyrosine phosphorylation of Cx43 dur-

ing mitosis) were observed by others using mitotic human

umbilical vein endothelial cells (3). Therefore, Src does not

A. interphase Rat-i B. Mitotic Rat-i

�: �0 -

4 *

L_*. pH 1.9 Buffer

C. Peptide D. Predicted Mobility

pSDPYHATTGPLSPSK

.3SDPYHAUGPLSPSKDCGSPK

DCGSPK

t�j. �


E. Mitotic Rat-ilPeptide Mix F. Mitotic Rat-i + Inhibitor

It; �

�. p

..*�p

t�j�

appear to have a direct role in Cx43 phosphorylation during

mitosis.

It is possible that loss of gjc during mitosis is due to loss

of gap junctions at the surface of the plasma membrane.

Previous studies reported a loss of gap junctions from theplasma membrane surface of proliferating hepatocytes in

regenerating liver tissue (43, 44). Mitosis-specific redistribu-

tion of Cx43 from the plasma membrane to intracellular

structures was observed in human umbilical vein endothelial

cells (3). Alternatively, phosphorylation of Cx43 during mito-

sis might trigger internalization and/or degradation of gapjunction channels, resulting in reduced gjc. Numerous pro-

Fig. 5. Tryptic phosphopeptide analysis of Cx43phosphorylated dunng interphase and mitosis in vivoand by p34cdc2 kinase in vitro. In vivo 32P-labeledCx43 proteins from Rat-i fibroblasts and a syntheticpeptide corresponding to amino acids 241-264 ofCx43 phosphorylated in vitro by purified p34��C2 wereisolated and digested with N-tosyl-L-phenylalaninechloromethyl ketone-trypsin. The resulting phos-phopeptides were separated by electrophoresis (pH1.9 buffer) in the horizontal dimension and chroma-tography (isobutyric acid buffer) in the vertical dimen-sion. Shown are 2-dimensional tryptic phosphopep-tide maps of: A, Cx43 from interphase cells; B, Cx43from nocodazole-arrested mitotic cells; C, syntheticpeptide phosphorylated by p34� in vitro; D, pre-dicted mobility of synthetic phosphopeptide frag-ments; E, mix of mitotic Cx43 and synthetic phos-phopeptidefragments; and F, Cx43 from mitotic cellsin the presence of butyrolactone I. The major phos-phopeptides observed during mitosis are numbered1-4. *, the origin.

teins are degraded by the ubiquitin-proteasome proteolyticpathway (reviewed in Ref. 45), including Cx43 (46). Recently,several groups demonstrated phosphorylation-dependentubiquitination and degradation of cyclins Di and E (47, 48).

Whether phosphorylation is required for ubiquitination and/or

degradation of Cx43 is not known.Phosphorylation of cellular substrates by p34cdc2 leads to

changes in cell structure during mitosis (reviewed in Refs.

16-18). Cell cycle progression through G2-M phase is de-

pendent on the phosphorylation of substrate proteins (re-viewed in Refs. 16-i 8). The substrates include nuclear pro-teins such as histone Hi , nuclear lamins, and nucleolin,


which upon phosphorylation by p34cdc2, induce mitotic-spe-cific changes in nuclear structure (49-54). Proteins important

for changes in cytoskeletal architecture during mitosis, in-cluding vimentin, plectin, caldesmon, and myosin II light

chain, also have been identified as substrates of p34�(55-63). p34cdc2mediated Cx43 phosphorylation and re-duced gap junctional permeability during mitosis may allowcell division to occur without interfering with the homeostasisof nonproliferating neighbors.

Cell cycle-dependent regulation of ion channels, includingpotassium and chloride channels, has been described (64,65). Recently, the effects of p3.4cdc2 kinase on potassium ionchannel function were reported (66). Mitotic-specific sup-pression of R-eag, a delayed rectifying potassium channel,was shown to be dependent on the presence of p34�2kinase/cyclin B activity. Although four p34�#{176}� phosphoryla-tion consensus sites were identified in the primary sequence

of R-eag, direct phosphorylation by p34cdc2 kinase was notexamined. The results described here add gap junctionchannels to an expanding list of pore-forming channelsphosphorylated and/or regulated by p34cdc2 during the cellcycle.

Materials and MethodsCell Culture and Synchronization. Rat-i fibroblasts (67) were culturedin DMEM supplemented with 10% calf serum (Intergen Co., Purchase,NY). Rat-i cells to be used as G0 cells were cultured in 0.5% calf serum

DMEM for 48 h prior to harvesting. Cells were synchronized at the G1-Sboundary by double thymidine block as described previously (68). Briefly,5 x 10� cells were blocked with 2 mpi thymidine for 14 h, released fromthe first block by three washes with PBS, then Incubated with mediumcontaining 24 � deoxycytidine and 24 �ai thymidine. After 9 h, the cellswere washed three times with PBS and Incubated with 2 m� thymidine for14 h. Following the second thymidine block, cells blocked at G1-S wereharvested. Cells to be collected at S and G2-M phases of the cell cyclewere released by three washes with PBS and incubated with 24 �u�i

deoxycytidine and 24 �ii4 thymidine. S-phase cells were harvested 4 hafter release from the second thyrnldine block. To block cells at G2-M,nocodazole (100 ng/ml) was added 6 h after second release and her-vested 4 h later. M-phase cells were obtained from either untreated ornocodazole-arrested mitotic cells by gentle pipethng and slow-speedcentrifugation. The attached cells (mainly in G2 phase) were harvestedfollowing removal of M-phase cells. To inhibit in vivo cdc2 activity duringG2-M, butyrolactone I (236 �i; a gift of Dr. Akira Okuyama, BanyuTsukuba Research Institute, Japan) was added 1 h prior to the addition ofnocodazole. Cell cycle stage was monitored by flow cytometry as do-scribed previously(69). Briefly, Rat-i fibroblasts (5 x i0� cells) were fixedwith 70% ethanol and stained with propidium iodide(40 �g/m� containingRNase (50 � The samples were analyzed for DNA content at G1, S,and G2-M on a Becton Dickinson FACScan.

Cell Lysls, Immunoprecipltatlon, arid Gel Electrophoresis. Expo-nentially growing or synchronized Rat-i fibroblasts were rinsed with coldPBS and lysed in 1% NP4O lysis buffer[20 mti Tris (pH 8.0), 150 m� NaCI,1 mM DTF, and 1 % NP4O]. The lysates were clarified by centrifugation and

normalized for protein content. Proteins were immunoprecipitated byincubating 1 ml of lysate containing 1 mg of protein with 1 �g of a rabbit

antiserum directed against a peptide encoding amino acids 368-382 ofrat heart Cx43 (18) or i �g of a cdc2 rabbit antiserum (70) for 2 h at 4#{176}Con a rocker. Repligen protein A beads (Repligen Corp., Cambridge, MA)were added, and the samples were incubated for an additional 2 h (acdc2IP) or 12 h (aCx43 IP). The immunoprecipitated Cx43 proteins wereextensively washed in lysis buffer and boiled in SDS sample buffer.cdc2/cyclin B complexes bound to beads were prepared form vitro kinaseassays. The proteins were resolved by SDS-PAGE on 12% (aCx43 IP) or20% (in vitro kinase reactions) polyacrylamide gels (71). Although the IPswere conducted using previously titered antibodies and cellular lysates

were normalized for protein content, the quantitative limitations of thisprocedure should be taken into account when interpreting the data.

lmmunoblotting. Proteins separated by SDS-PAGE as described

above were electrotransfen’ed to Immobilon polyvinylidene difluoridemembrane (Millipore, Bedford, MA) and blocked in 3% BSA blocking

buffer [3% (w/v) BSA-RIA grade, 50 m�i Tns (pH 7.5), 150 m� NaCI, and0.1 % Tween-20] overnight at room temperature on a rocker. The mern-brane was probed with aCx43 antibody followed by a horseradish per-oxidase-coupled secondary antibody. The immunoreactive proteins were

detected by enhanced chemiluminescence (Amersham Corp., MingtonHeights, IL).

PAP Treatment. Dephosphorylation of Cx43 proteins with PAP wasconducted as described previously (72). Briefly, Cx43 immunoprecipitates

were washed three times with RIPA lysis buffer lacking SDS and deoxy-

cholate [150 mM NaCI, 1% Triton X-iOO, 10 m� Tris(pH 7.2), i m� sodiumorthovanadate, 1 mM sodium fluoride, 0.5 m�.i phenylmethylsulfonyl flue-

ride, 20 pg/mI leupeptmn, and 1 �g/ml aprotinin] and twice In phosphatasebuffer [20 mM 2-N-morpholino ethanesulfonic acid (pH 5.5), 1 m� MgCI2,

0.1 mM DTT, 4 �g/mlleupeptin, 2% aprotinin, and 4 �ig/ml soybean trypsininhibitor]. The samples were incubated with 0.2 unit PAP for 60 mm at

37#{176}C,washed threetimes with RIPAlysis buffer, and boiled in SDS samplebuffer. The samples were resolved by SDS-PAGE and immunoblotted

with aCx43 as described above.

MetabolIc Labeling. Exponentially growing or synchronized Rat-i fi-

broblasts were incubated in phosphate-free medium containing 0.5% (G�)or 4% (Asyn, G1-S, 5, and G2-M) dialyzed calf serum for 1 h at 37#{176}C,followed by incubation with 1 mCVml of [�2P]P� (ICN, Irvine, CA) for 4 h at

37#{176}C.For cells arrested at G1-S, the labeling media contained 2 m�thymidine. For cells synchronized at S phase, the labeling media con-

tamed 24 �LM deoxycytidmne and 24 � thymidine. For cells arrested at

G2-M, the labeling medium contained 24 p.�.i deoxycytidmne, 24 �tM thymi-dine, and 100 ng/ml nocodazole in the presence or absence of 236 pii

butyrolactone I. Following the labeling period, the cells were rinsed withcold PBS, solubilized in RIPA lysis buffer, and clarified by centrifugation.

Cx43 proteins were immunoprecipitated and resolved by SDS-PAGE asdescrIbed above. 32P-iabeled Cx43 proteins were quantified by gel puri-fication followed by Cerenkov counting in a Beckman LS 6000 SC spec-trometer (Beckman Instruments, Fullerton, CA).

In Vltm Kinase Assay. Purified cdc2/cyclmn B4 or cdc2/cyclmn B com-plex immunoprecipitated from cell lysates of Rat-i fibroblasts, as do-scnbed above, were used to phosphorylate histone Hi , GST Cx43 CT tail(amino acids 236-282; Ref. 19), or a synthetic peptide corresponding toamino acids 241-264 of Cx43 in vitro. cdc2 immunoprecipitates werewashed four times in I % NP4O lysis buffer and twice in kinase assay

buffer minus Mg�2 and [�y-32P]ATP. The kinase reactions were performedin 20-s.d reaction volumes containing 50 m� HEPES (pH 7.4), 10 m�MgCl2, 1 �M DTT, 25 �LM ATP, 10 �Ci [y-32PJ ATP, and 1 �g substrate in

the presence or absence of 62 butyrolactone I. The reactions wereInitiated by the addition of purified cdc2/cyclin B (1 p.g) or cdc2 immuno-

precipitates and incubated for 30 mm at 30#{176}C.The reactions were termi-

nated by the addition of SDS sample buffer, and the phosphorylatedsubstrates were resolved by SDS-PAGE on i2% (histone Hi and GSTCx43 tail) or 20% (synthetic peptide) polyacrylamide gels as described

above.Two-DImensional Tryptlc Phosphopeptide MappIng and Phos-

phoamino Acid Analysis. Two-dimensional analysis of tryptic phos-phopeptides was performed as described previously (25). BrIefly, in vivo�P-labeled Cx43 proteins from Rat-i fibroblasts were gel purified, eluted,precipitated with trlchloroacetic acid and oxidized in performic acid. Thephosphorylated synthetic peptide was separated on cellulose TLC plates(E. Merck; 100 g.�m) by electrophoresis using pH 3.5 buffer [5.0% (vlv)glacial acetic acid and 0.5% (v/v) pyridine]for30 mm at 1 .5 kV and purified

from the cellulose by eluting with pH 1 .9 buffer [2.5% (v/v) formic acid

(88%) and 7.8% (v/v) glacial acetic acid]. The samples were digested with25 �g of N-tosyl-L-phenylalanine chloromethyl ketone-trypsin (Worthing-ton, Freehold, NJ), and the resulting tryptic phosphopeptides were sep-

arated on cellulose TLC plates by electrophoresis using pH 1.9 buffer for22 mm at 1 .5 kV in the first dimension and by ascending chromatography

using isobutync acid buffer [62.5% (v/v) isobutyric acid, 1 .9% (v/v)n-butanol, 4.8% (v/v) pyridmne, and 2.9% (v/v) glacial acetic acid] in the

second dimension. The phosphopeptides were detected by exposure to


18. Moreno, S., and Nurse, P. Substrates for p34�: in vivo veritas? Cell,

61: 549-551, i990.

Kodak X-OMAT XAR-5 film with an intensifying screen (Dupont,Wilmington, DE) at -70#{176}C.

Phosphoamino acid composition was determined as described previ-ously (25). Briefly, 32P-iabeled Cx43 proteins were hydrolyzed in 6 N HCLfor i h at i iO#{176}C.The samples were lyophilized, resuspended in pH i .9buffer containing i �g each of phosphoserine, phosphothreonine, andphosphotyrosine, and resolved on TLC plates by two-dimensional alec-

trophoresis using pH i .9 buffer for 20 mm at i .5 kV in the first dimension

and pH 3.5 buffer [5.0% (v/v) glacial acetic acid and 0.5% (v/v) pyridine]for i6 mm at i .3 kV in the second dimension. The phosphoarnino acidstandards were visualized with nmnhydrin, and the TLC plates were auto-radiographed as described above.

AcknowledgmentsWe thank Dr. Akira Okuyama, Banyu Tsukuba Research Institute, Japan,

for generously providing butyrolactone I; Dr. Alan F. Lau for helpful dis-cussions; and Jill Meisenhelder, Suzanne Simon, and Helen Mondala fortechnical assistance.

References1 . Norbury, C., and Nurse, P. Animal cell cycles and their control. Annu.Rev. Biochem., 61: 44i-470, i992.

2. Stein, L S., Boonstra, J.. and Burghardt, R. C. Reduced cell-cellcommunication between mitotic and nonmitotic coupled cells. Exp. CellRes., 198: i-7, 1992.

3. Xie, H., Laird, D. W., Chang, T., and Hu, V. W. A mitosis-specificphosphorylation of the gapjunction protein connexin43 in human vascular

cells: biochemical characterization and localization. J. Cell Biol., 137:203-2i0, i997.

4. Miller, S. M., Garfield, R. E., and Daniel, E. E Improved propagation inmyometrium associated with gap junctions during parturition. Am. J.Physiol., 256: Ci30-Ci4i , i989.

5. Spray, D. C., and Burt, J. M. Structure-activity relations of the cardiacgap junction channel. Am. J. Physiol., 258: i95-205, i990.

6. Furshpan, E. J., and Potter, D. D. Transmission at the giant motorsynapses of the crayfish. J. Physiol., 145: 289-325, 1959.

7. Warner, A. E., Guthrie, S. C., and Gilula, N. B. Antibodies to gap-junctional protein selectively disrupt junctional communication in the earlyamphibian embryo. Nature (Lond.), 31 1: 127-i31 , 1984.

8. Caveney, S. The role of gap junctions in development. Annu. Rev.

Physiol., 47: 319-335, i985.

9. Loewenstein, W. R., and Rose, B. The cell-cell channel in the control ofgrowth. Semin. Cell Biol., 3: 59-79, i992.

io. Yamasaki, H., and Naus, C. C. G. Role of connexin genes in growthcontrol. Carcinogenesis (Lond.), 17: i 199-1213, 1996.

1 i . Goodenough, D. A., Goliger, J. A., and Paul, D. L Connexins, con-nexons, and intercellular communication. Annu. Rev. Blochem., 65: 475-

502, i996.

i2. Bennett, M. V. L, Barrio, L C., Bargiello, T. A., Spray, D. C.,Hertzberg, E, and Saez, J. C. Gapjunctions: new tools, new answers, newquestions. Neuron, 6: 305-320, 1991.

i3. Lau, A. F., Kurata. W. E.. Kanernitsu, M. V.. Loo, L W. M., Warn-Cramer, B. J., Eckhart, W., and Lampe, P. D. Regulation of connexin43

function by activated tyrosine protein kinases. J. Bioenerg. Biomemb., 28:359-368, 1996.

i4. Yeager, M., and Nicholson, B. J. Structure of gapjunctlon intercellularchannels. Curr. Opin. Struct. Biol., 6: 183-192, i996.

15. Bruzzone, R., White, T. W., and Paul, D. L Connections with oonnex-ins: the molecular basis of direct intercellular signalling. Eur. J. BiOChem.,238: 1-27, i996.

i6. Nigg, E. A. Cellular substrates of p34� and its companion cyclin-dependent kinases. Trends Cell Biol., 3: 296-301 , 1993.

17. Lewin, B. Driving the cell cycle: M phase kinase, its partners, andsubstrates. Cell, 61: 743-752, 1990.

i9. Crow, D. S., Beyer, E. C., Paul, D. C., Kobe, S. S., and Lau, A. F.Phosphorylation of oonnexin43 gap junction protein in uninfected andRous sarcoma virus-transformed mammalian fibroblasts. Mol. Cell. Biol.,10: 1754-1763, 1990.

20. Loo, L W. M., Berestecky, J. M.. Kanemitsu, M. V., and Lau, A. F.pp60�-mediated phosphorylation of connexin43, a gapjunction protein.J. Biol. Chem., 270: i275i-i2761, 1995.

21 . Musil, L S., Cunningham, B. A., Edelman, G. M., and Goodenough,D. A. Differential phosphorylation of the gap junction protein connexin43in junctional communication-competent and -deficient cell lines. J. CellBiol., 111: 2077-2088, i990.

22. Kitagawa, M., Okabe, T., Ogino, H., Matsumoto, H., Suzuki-Takahashi, I., Kokubo, T., Higashi. H., Saitoh, S., Taya, V., Yasuda, H.,Ohba, V., Nishimura, S., Tanaka, N., and Okuyama, A. Butyrolactone I, aselective inhibitor of cdk2 and cdc2 kinase. Oricogene, 8: 2425-2432,1993.

23. Kitagawa, M., Higashi, H., Takahashi, I. S., Okabe, T., Ogino, H., Taya,V., Nishimura, S., and Okuyama, A. A cyclin-dependent kinase inhibitor,butyrolactone I, inhibits phosphorylation of RB protein and cell cycleprogression. Oncogene, 9: 2549-2557, 1994.

24. Meijer, L Chemical inhibitors of cyclin-dependent kinases. Trends

Cell Biol., 6: 393-397, 1996.

25. Boyle, W. J., van der Gear, P., and Hunter, T. Phosphopeptide map-ping and phosphoamino acid analysis by two-dimensional separation on

thin-layer cellulose plates. Methods Enzymol., 201: i iO-i49, i99i.

26. Trosko. J. E., Chang, C. C., Madhykar, B. V., Oh, S. V., Bombick, D.,and el Fouly, M. H. Modulation ofgapjunction intercellular communication

by tumor promoting chemicals, oncogenes and growth factors duringcarcinogenesis. In: E. L Hertzberg and R. G. Johnson (eds.), Gap Juno-tions, pp. 435-448. New York: Alan R. Use, Inc., 1988.

27. Do Mello, W. C. Cyclic nucleotides and junctional permeability. In:E. L Hertzberg and R. G. Johnson (eds.), Gap Junctions, pp. 245-254.New York: ,�Jan R. Use, Inc., i988.

28. Kanemitsu, M. Y., and Lau, A. F. Epidermal growth factor stimulatesthe disruption of gap junctional communication and connexin43 phos-phorylation independent of i2-O-tetradecanoylphorbol i3-acetate-sensi-tive protein kinase C: the possible involvement of mitogen-activated pro-tein kinase. Mol. Biol. Cell, 4: 837-848, 1993.

29. Marchiori, F., Megglo, F., Mann, 0., Born, G., Calderan, A., Ruzza, P.,and Pinna, L A. Synthetic peptide substrates for casein kinase 2. Assess-mont of minimum structural requirements for phosphorylatlon. Biochim.Biophys. Acts, 971: 332-338, 1988.

30. Kuenzel, E. &, Mulligan, J. A., Sommercorn, J., and Krebs, E G.Substrate specificity determinants for casein kinase II as deduced fromstudies with synthetic peptides. J. Biol. Chem., 262: 9136-9140, 1987.

31 . Litchfleld, D. W., Luscher, B., Lozeman, F. J., Eisenman, A. N., and

Krebs, E G. Phosphorylation of casein kinase II by p34�2 in vitro and atmitosis. J. Biol. Chem., 267: 13943-13951, 1992.

32. Abrieu, A., Doree, M., and Picard, A. Mitogen-activated protein kinaseactivation down-regulates a mechanism that inactivates cyclin B-cdc2

kinase in G2-arrested oocytes. Mol. Biol. Cell, 8: 249-261, 1997.

33. Warn-Cramer, B. J., Lampe, P. D., Kurata, W. E., Kanemitsu, M. V.,Loo, L W. M., and Lau, A. F. Characterization of the mitogen-activatedprotein kinase phosphorylation sites on the connexin-43 gap junctionprotein. J. Blol. Chem., 271: 3779-3786, i996.

34. Chackalaparampil, I., and Shalloway, D. Altered phosphorylation andactivation of pp60c�arc during fibroblast mitosis. Cell, 52: 801-810, 1988.

35. Shenoy, S., Choi, J-K., Bagrodia, S., Copeland, T. D., Mailer, J. L,and Shalloway, D. Purified maturation promoting factor phosphorylatespp6Oc-&c at the sites phosphorylated during fibroblast mitosis. Cell, 57:763-774, 1989.

36. Shenoy, S., Chackalaparampil, I., Bagrodia, S., Lin, P-H., and

Shalloway, D. Role of p34�2-mediated phosphorylations in two-stepactivation of pp6Oc-&c during mitosis. Proc. NatI. Acad. Sci. USA, 89:7237-7241, 1992.

37. Ralston, R., and Bishop, J. M. The product of the protooncogenec-sm is modified during the cellular response to platelet-derived growthfactor. Proc. NatI. Acad. Sci. USA. 82: 7845-7849, 1985.


38. Gould, K. L, and Hunter, T. Platelet-derived growth factor inducesmultisite phosphorylation of pp60’�� and increases its protein-tyrosmnekinase activity. Mol. Cell. Biol., 8: 3345-3356, i988.

39. Kypta, R. M., Goldberg, V., Ulug, E. T., and Courtneidge, S. A.Association between the PDGF receptor and members ofthe sit family oftyrosine kinases. Cell, 62: 481-492, i990.

40. Roche, S., Fumagalli, S., and Courtneidge, S. A. Requirement for Srcfamily protein tyrosine kinases in G2 for fibroblast cell division. Science(Washington DC), 269: i567-i569, i995.

4i . Crow, D. S., Kurata, W. E., and Lau, A. F. Phosphorylation of con-nexin43 in cells containing mutant sit oncogenes. Oncogene, 7: 999-1003, 1992.

42. Swenson, K I., Piwnica-Worms, H., McNamee, H., and Paul, D. LTyrosine phosphorylation of the gap junction protein connexin43 is re-

quired for the pp�()V�S�Cind� inhibition of communication. Cell Regul.,1: 989-1002, i990.

43. Traub, 0., Druge, P. M., and Willecke, K Degradation and resynthesis

of gap junction protein in plasma membranes of regenerating liver afterpartial hepatectomy or cholestasis. Proc. NatI. Acad. Sd. USA, 80: 755-

759, i983.

44. Dermietzel, R., Yancey, S. B., Traub, 0., Willecke, K., and Revel, J-P.Major loss of the 28-kD protein of gap junction in proliferating hepato-

cytes. J. Cell Biol., 105: i925-i934, 1987.

45. Ciechanover, A. The ubiquitin-proteasome proteolytic pathway. Cell,79: i3-2i, 1994.

46. Uang, J. G., and Beyers, E. C. The gapjunction protein connexin43 isdegraded via the ubiquitin proteasome pathway. J. Biol. Chem., 270:26399-26403, 1995.

47. Won, K, and Reed, S. I. Activation of cyclin E/CDK2 is coupled tosite-specific autophosphorylation and ubiquitin-dependent degradation

of cyclin E. EMBO J., 15: 4182-4193, 1996.

48. Diehl, J. A., Zindy, F., and Sherr, C. J. Inhibition of cyclin Di phos-phorylation on threonine-286 prevents its rapid degradation via the ubiq-uitin-proteasome pathway. Genes Dev., 11: 957-972, 1997.

49. Anon, D., Meijer, L, Brizuela, L, and Beach, D. cdc2 is a component

ofthe M phase-specific histone Hi kinase: evidence foridentity with MPF.Cell, 55: 371-378, i988.

50. Luscher, B., Brizuela, L, Beach, D., and Eisenman, R. N. A role for thep34cdc2 � and phosphatases in the regulation of phosphorylation anddisassembly of lamin B2 during the cell cycle. EMBO J., 10: 865-875,1991.

51. Peter, M., Nakagawa, J., Doree, M., Labbe, J. C., and Nugg, E. A. Invitro disassembly of the nuclear lamina and M phase-specific phospho-rylatlon of lamins by cdc2 kinase. Cell, 61: 591-602, 1990.

52. Dessev, G., lovcheva-Dessev, C., Bischoff, J. R., Beach, D., andGoldman, R. A complex containing p34� and cyclin B phosphorylatesthe nuclear lamin and disassembles nuclei of clam oocytes in vitro. J. CellBiol., 112: 523-533, 1991.

53. Peter, M., Nakagawa, J., Doree, M., Labbe, J. C., and Nigg, E. A.Identification of major nucleolar proteins as candidate mitotic substratesof cdc2 kinase. Cell, 60: 791-801 , 1990.

54. Belenguer, P., Caizergues-Ferrer, M., Labbe, J-C., Doree, M., andAmalric, F. Mitosis-specific phosphorylation of nucleolin by p34� pro-ten kinase. Mol. Cell. Biol., 10: 3607-3618, 1990.

55. Chou, V-H., Bischoff, J. R., Beach, D., and Goldman, R. D. Interme-

diate filament reorganization during mitosis is mediated by p34� phos-phorylation of vimentin. Cell, 62: 1063-1071, 1990.

56. Chou, V-H., Rosevear, E., and Goldman, R. D. Phosphorylation anddisassembly of Intermediate filaments in mitotic cells. Proc. NatI. Acad.Sd. USA, 86: 1885-1889, 1989.

57. Malecz, N., Foisner, R., Stadler, C., and Wiche, G. Identification ofplectin as a substrate of p34� kinase and mapping of a single phos-phorylation site. J. Biol. Chem., 271: 8203-8208, 1996.

58. Evans, R. M., and Fink, L M. An alteration in the phosphorylation ofvimentin-type intermediatefilaments is associated with mitosis in culturedmammalian cells. Cell, 29: 43-52, 1982.

59. Yamashiro, S., Vamakita, V., Ishikawa, R., and Matsumura, F. Mitosis-

specific phosphorylation causes 83K non-muscle caldesmon to dissoci-ate from microfllaments. Nature (Lond.), 344: 675-678, 1990.

60. Yamashlro, S., Vamakita, V., Hosoya, H., and Matsumura, F. Phos-phorylatlon of non-muscle caldesmon by p34� kinase during mitosis.Nature (Lond.), 349: 169-172, 1991.

6i . Satterwhite, L L, Lohka, M. J., Wilson, K. L, Scherson, T. V., Cisek,L J., Corden, J. L, and Pollard, T. D. Phosphorylation of myosin-Ilregulatory light chain by cyclmn-p34�: a mechanism for the timing ofcytokinesis. J. Cell Biol., 118: 595-605, i992.

62. Hosoya, H., Yamashiro, S., and Matsumura, F. Mitosis-specific phos-phorylation of myosin light chain kinase. J. Biol. Chem., 266: 22173-

22178, 1991.

63. Yamakita, V., Yamashiro, S., and Matsumura, F. In vivo phosphoryl-ation of regulatory light chain of myosin II during mitosis of cultured cells.J. Cell Biol., 124: 129-137, 1994.

64. Block, M. L, and Moody, W. J. A voltage-dependent chloride currentlinked to the cell cycle in ascidian embryos. Science (Washington DC),,

247: i090-i092, 1990.

65. Day, M. L, Pickering, S. J., Johnson, M. H., and Cook, D. I. Cell-cyclecontrol of a large-conductance K� channel in mouse eariy embryos.Nature (Lond.), 365: 560-562, 1993.

66. Bruggemann, A., Stuhmer, W., and Pardo, L A. Mitosis-promotingfactor-mediated suppression of a cloned delayed rectifier potassiumchannel expressed in Xenopus oocytes. Proc. NatI. Acad. Sci. USA, 94:

537-542, 1997.

67. Reynolds, A. B., Vila, J., Lansing, T. J., Potts, W. M., Weber, M. J., andParsons, J. T. Activation ofthe oncogenic potential ofthe avian cellularsrcprotein by specific structural alteration ofthe carboxy terminus. EMBO J..6: 2359-2364, 1987.

68. Heintz, N., Sive, H. L, and Roeder, A. G. Regulation of human histonegene expression: kinetics of accumulation and changes in the rate ofsynthesis and in the half-lives of individual histone mRNAs during theHeLa cell cycle. Mol. Cell. Biol., 3: 539-550, 1983.

69. Jiang, W., Kahn, S. M., Zhou, P., Zhang, V. J., Cacace, A. M., Infante,A. S., Dol, S., Santella, A. M., and Weinstein, I. B. Overexpression of cyclmnDi in rat fibroblasts causes abnormalities in growth control, cell cycleprogression and gene expression. Oncogene, 8: 3447-3457, 1993.

70. Watanabe, N., Broome, M., and Hunter, T. Regulation of the WEE1 Huhuman CDK tyrosine-15 kinase during the cell cycle. EMBO J., 14: 1878-1891, 1995.

71 . Laemmli, U. K Cleavage of StrUCtUral proteins during the assembly ofthe head bacteriophage T4. Nature (Lond.), 227: 680-685, 1970.

72. Meisenhelder, J., and Hunter, T. Methods for studying the phospho-rylation of phospholipase C in vivo and in vitro. Methods Enzymol., 197:288-305, 1991.

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