©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
pp60 -mediated Phosphorylation of Connexin 43, a Gap Junction Protein (*)

Lenora W. M. Loo (1), John M. Berestecky (2), Martha Y. Kanemitsu (1)(§), Alan F. Lau (1)(¶)

From the (1) Molecular Carcinogenesis Program, Cancer Research Center of Hawaii and Department of Genetics and Molecular Biology, School of Medicine, and the (2) Mathematics-Science Department, Kapiolani Community College, University of Hawaii at Manoa, Honolulu, Hawaii 96813

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Several laboratories have demonstrated a decrease in gap junctional communication in cells transformed by the src oncogene of the Rous sarcoma virus. The decrease in gap junctional communication was associated with tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). This study was initiated to determine if the phosphorylation of Cx43 is the result of a direct kinase-substrate interaction between the highly active tyrosine kinase, pp60, and Cx43. Previous biochemical studies have been limited by the low levels of Cx43 protein in fibroblast cell lines. To obtain larger quantities of Cx43, we constructed a recombinant baculovirus expressing Cx43 in Spodoptera frugiperda (Sf-9) cells and subsequently purified the expressed Cx43 by immunoaffinity chromatography. We observed that this partially purified Cx43 was phosphorylated on tyrosine in vitro in the presence of kinase-active pp60. Phosphotryptic peptide mapping indicated that the in vitro phosphorylated Cx43 contained phosphopeptides which comigrated with a subset of tryptic peptides prepared from Cx43 phosphorylated in vivo. Furthermore, coinfection of Sf-9 cells with recombinant baculoviruses encoding pp60 and Cx43 resulted in the accumulation of phosphotyrosine in Cx43. Taken together, the evidence presented in this paper demonstrates that kinase active pp60 is capable of phosphorylating Cx43 in a direct manner. Since the presence of phosphotyrosine on Cx43 is correlated with the down-regulation of gap-junctional communication, these results suggest that pp60 regulates gap junctional gating activity via tyrosine phosphorylation of Cx43.


INTRODUCTION

Gap junctions, which are found in plasma membranes of almost all animal cells, mediate cell-to-cell communication and maintain normal organ function (1) , embryonic development (2) , and perhaps growth control (3, 4, 5, 6, 7) . Gap junctions are formed when a connexon (hemichannel) from one cell docks with a symmetrically opposed connexon from an adjacent cell forming a conduit which permits the passive diffusion of ions and small molecules (<1000 daltons) between neighboring cells. Each connexon consists of an oligomer of six connexin proteins that form the central aqueous pore (8) .

Connexins are a family of gap junction proteins that are composed of four transmembrane domains, two extracellular loops, an intracellular loop, and amino- and carboxyl-terminal domains located in the cytoplasm (9, 10, 11) . The transmembrane domains and extracellular loops are highly conserved between family members and are believed to be responsible for channel structure, whereas the cytoplasmic domains are less conserved and may account for the differential regulation of gap junctions (12) .

Gap junctional communication (GJC)() has been implicated in the regulation of growth control based on experiments linking the permeability of the gap junction channel with cellular transformation (reviewed in Ref. 4). Several groups have demonstrated that the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), can inhibit GJC (13, 14). Transformation of cells by viral oncogenes such as v-ras(15, 16) , v-src(17, 18) , v-mos (19), and polyomavirus middle T antigen (20) has been correlated with an inhibition of GJC. Furthermore, Mehta et al.(21) demonstrated that treatment of chemically transformed CH3 10T1/2 cells with retinoids simultaneously increased GJC and inhibited transformation. The most direct evidence supporting the role of GJC in transformation involved the introduction of connexin 43 (Cx43) into communication-deficient, carcinogen-transformed cells. Re-expression of Cx43 resulted in restoration of GJC, an inhibition of growth, focus formation (5) , and tumorigenicity (22) . Similar results were observed in communication-deficient C6 glioma cells transfected with Cx43 (23) and human tumor cells (SKHep) transfected with Cx32 (24) .

The permeability of the gap junction channel is affected by various factors such as changes in free intracellular calcium levels (25) , intracellular pH (26) , voltage (27) , growth factors (28, 29) , and transforming oncogene activity (19, 30) . The mechanisms that mediate changes in gap junctional permeability are at present incompletely understood. However, the increased phosphorylation of connexin induced by various stimuli is associated with modulation of channel gating (opening and closing) (31, 32) . For example, cyclic AMP treatment of liver hepatocytes increased GJC and connexin 32 (Cx32) phosphorylation, which may be the result of activated cyclic AMP-dependent serine kinases (33, 34) . Expression of the ras oncogene or TPA treatment of mouse primary keratinocytes decreased GJC and increased serine phosphorylation on Cx43 (15) . Epidermal growth factor treatment of T51B rat liver epithelial cells produced a rapid, transient decrease in GJC, which was associated with increased serine phosphorylation on Cx43 (28, 35) .

RSV possesses the oncogene v-src, which encodes a plasma membrane-associated tyrosine kinase, pp60 (36, 37). Expression of pp60 results in a loss of growth control and cellular transformation. Tyrosine phosphorylation on Cx43 in v-src-transformed cells correlates with a decrease in GJC (38, 39, 40) . Cells infected with a temperature-sensitive mutant of RSV demonstrated a rapid reduction in GJC and a transformed morphology when shifted to the permissive temperature, where pp60 is active. At the nonpermissive temperature, where pp60 is inactive, the cells exhibited high levels of GJC and a normal morphology (18, 30) . Phosphorylated Cx43, purified from cells shifted to the permissive temperature, rapidly accumulated phosphotyrosine (Tyr(P)), concomitant with the disruption of GJC (39) . In contrast, cells grown at the nonpermissive temperature, with normal levels of GJC contained Cx43 phosphorylated only on phosphoserine (Ser(P)) residues. These results indicated that pp60 may decrease GJC by inducing the phosphorylation of Cx43 on tyrosine.

Further evidence for this proposed mechanism came from experiments utilizing Xenopus oocytes expressing Cx43 and/or pp60. When Cx43 was coexpressed with pp60, junctional conductance was disrupted and Cx43 was phosphorylated on tyrosine. Oocytes coexpressing a mutated form of Cx43 (tyrosine 265 mutated to phenylalanine) and pp60 did not demonstrate a decrease in GJC or tyrosine phosphorylation on Cx43 (41) . Although these results suggested that pp60 may directly regulate channel gating via tyrosine phosphorylation of Cx43 on residue 265, it is possible that these events in the oocytes do not completely reflect the events in mammalian cells.

pp60 may activate a secondary tyrosine kinase, which then phosphorylates Cx43. One such tyrosine kinase could be the focal adhesion kinase, pp125(42) , which is phosphorylated on tyrosine, activated in v-src-transformed cells (43) , and associated with pp60(44) .

To test the hypothesis that Cx43 is a substrate of activated pp60, we examined the ability of purified pp60 to phosphorylate either whole Cx43 purified from baculovirus-infected insect cells or the carboxyl tail region of Cx43 fused to glutathione S-transferase (GST). In addition, we examined the phosphorylation of Cx43 coexpressed with pp60 in insect cells. The results from this study indicated that Cx43 can serve as a substrate of activated pp60 in in vitro kinase reactions and in intact mammalian and insect cells. Furthermore, these data strongly suggest that the tyrosine residue(s) phosphorylated by pp60 are located in the carboxyl tail region of Cx43. Since tyrosine phosphorylation on Cx43 has been correlated with a down-regulation in GJC, these results suggest that activated pp60 has a direct role in modulating the gating of gap junctions.


MATERIALS AND METHODS

Cell Culture

Sf-9 insect cells (Invitrogen) were grown as monolayers in Grace's insect medium (Life Technologies, Inc.) supplemented with 3.3 g/liter yeastolate (Life Technologies, Inc.), 3.3 g/liter lactalbumin hydrolysate (Life Technologies, Inc.) and 10% fetal calf serum (HyClone) at 27 °C. Sf-9 cells were infected with recombinant virus at a multiplicity of infection of 5-10 and harvested 48-72 h post-infection. For coinfection experiments, cells were infected at a ratio of 1:2 for the recombinant pp60 and Cx43 baculoviruses, respectively. Rat-1 and v-src-transformed Rat-1 fibroblasts were grown as described previously (45) .

Construction of Recombinant Transfer Vector and Baculovirus

pVL1393-Cx43 was constructed from the transfer vector pVL1393 (Pharmingen) (46) and the Cx43 cDNA (G2 clone) (47) contained in Bluescript (Stratagene). The Bluescript-G2 construct was digested with BanI (New England Biolabs) and the 1.8-kilobase pair BanI fragment containing the Cx43 cDNA was blunt-ended with Klenow (Promega), then digested with EcoRI (U. S. Biochemical Corp.). This fragment was then inserted into a SmaI-EcoRI site of pVL1393 transfer vector resulting in the recombinant transfer plasmid, pVL1393-Cx43.

Recombinant baculovirus was generated by cotransfecting a monolayer of Sf-9 cells with recombinant transfer plasmid and BaculoGold baculovirus DNA (Pharmingen). Recombinant baculoviruses were then subjected to one round of plaque purification.

Western Blot Analysis

Expression of Cx43 in Sf-9 cells was examined by harvesting cells at specified time points. Cells were then lysed in 200 µl of lysis buffer (100 mM NaCl, 10 mM Tris (pH 8.0), 2 mM EDTA, 1% Triton X-100), and clarified in a Beckman TL-100 ultracentrifuge at 400,000 g for 20 min at 4 °C.

Samples for the biochemical subcellular localization experiments were obtained by lysing recombinant baculovirus-infected cells with cytosol extraction buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 2 mM EDTA (pH 7.5), 1 mM EGTA, 25 µg/ml leupeptin, and 25 µg/ml aprotinin) in a Dounce homogenizer. The samples were centrifuged at 131,000 g for 20 min at 4 °C in a Beckman TL-100 ultracentrifuge. The supernatant was collected and stored as the cytosolic fraction. The pellet was resuspended by passage through a 22-gauge, 3/8-inch needle five times in cytosol extraction buffer supplemented with 1% Triton X-100, followed by centrifugation at 400,000 g for 20 min at 4 °C in a Beckman TL-100 ultracentrifuge. The supernatant was collected and stored as the particulate fraction.

Protein concentrations were measured with the Bio-Rad protein assay kit. Whole and fractionated cell samples were boiled in an equal volume of 2 sample buffer (to achieve a final concentration of 2% SDS, 5% -mercaptoethanol, 5% glycerol, 0.004% bromphenol blue, and 0.03 M Tris (pH 6.8)), separated on 12% SDS-polyacrylamide gels (48) , and electrotransfered to Immobilon-P membranes (Millipore Corp.). The membranes were blocked overnight with blocking buffer (25 mM Tris-buffered saline (pH 7.4), 1% bovine serum albumin), washed 30 min with wash buffer (25 mM Tris-buffered saline (pH 7.4), 0.05% Tween 20, 1 mM EDTA), and incubated with rabbit antiserum directed against peptide 368-382 of Cx43 (anti-CT 368; 1:500 dilution) for 2 h at room temperature. The membranes were then washed for 45 min in wash buffer, incubated with 1 µCi of I-labeled goat anti-rabbit IgG (ICN Pharmaceuticals, Inc.) for 1 h at room temperature, and washed for 30 min. Membranes were then autoradiographed using Kodak X-Omat XAR-5 film with the aid of an intensifying screen at -70 °C. Immunoreactive bands were excised and quantitated with a Micromedics four-channel counter.

Immunofluorescence Microscopy

Sf-9 cells were infected as described above and harvested 48 h post-infection. Cells were fixed in 3% paraformaldehyde/PBS for 20 min at 4 °C, washed two times in PBS, permeabilized in 0.2% Triton X-100/PBS for 2 min at 4 °C, and washed two times in PBS. Cells were incubated with Cx43 rabbit antiserum (anti-CT 368; 1:500 dilution) overnight at 4 °C, washed two times, and blocked with normal goat serum (1:50 dilution) for 30 min at 4 °C. After washing twice with PBS, the cells were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (1:80 dilution) (Sigma) for 1 h at 4 °C and washed three times in PBS. Cells were wet-mounted in 50% PBS, 50% glycerol containing 1 mg/ml p-phenylenediamine (Sigma) and visualized and photographed with a Zeiss Axioplane Universal microscope equipped with epifluorescence.

Radiolabeling, Immunoprecipitation, and Quantitation of Cx43

Sf-9 cells were infected with recombinant baculovirus encoding Cx43 as described above. At 24 h post-infection, the cells were radiolabeled with either EXPRESS protein labeling mix (NEG-072; DuPont NEN) at 100 µCi/ml in Grace's methionine-free medium (Life Technologies, Inc.) or [P ] (NEX-053; DuPont NEN) at 0.5 mCi/ml in Grace's phosphate-deficient medium (Cell Culture Facility, University of California, San Francisco) supplemented with 0.4% fetal calf serum, 5% complete TNM-FH medium, 20 µM MES (pH 6.1) (Sigma). Cells were labeled overnight (16 h) at 27 °C and harvested. Fibroblasts were grown to confluence and labeled with EXPRESS protein labeling mix at 100 µCi/ml or [P ] at 0.5 mCi/ml in phosphate-deficient medium for 3 h at 37 °C. The cells were lysed in RIPA buffer (150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, 10 mM Tris (pH 7.2), 50 mM NaF, 160 µM NaVO, 1 mM phenylmethylsulfonyl fluoride), clarified, and immunoprecipitated with normal rabbit serum, rabbit serum directed against a Cx43 (CT368), or monoclonal antibody directed against pp60 (mAb 327, generously provided by Joan Brugge; 54) as described previously (38) . Immunoprecipitated proteins were resolved on a 7.5-15% SDS-polyacrylamide gradient gel. Gels were dried and autoradiographed using Kodak X-Omat XAR-5 film at -70 °C. Gels containing [S]-labeled proteins were fluorographed prior to drying. Gel pieces containing S- or P-labeled Cx43 proteins were excised, rehydrated, and counted in 7.5% Scintigest (New England Nuclear)-Scintiverse fluid (Fisher Scientific) using a Beckman LS5000 liquid scintillation counter.

Phosphoamino Acid Analysis

P-Labeled Cx43 was immunoprecipitated and gel-purified as described. The proteins were electrotransfered to an Immobilon-P membrane (Millipore) and acid-hydrolyzed. The phosphopeptides were resolved on thin-layer cellulose plates in two dimensions at pH 1.9 and 3.5, as described previously in Ref. 49. Autoradiography was performed with Kodak X-Omat XAR-5 film with the aid of an intensifying screen at -70 °C.

Preparation of Cx43 Monoclonal Antibodies and Immunoaffinity Matrix

Monoclonal antibodies were made according to established techniques (50) against a synthetic peptide corresponding to amino acids 241-254 of the COOH-terminal cytoplasmic domain of Cx43. The peptide was conjugated to rabbit serum albumin via an amino-terminal cysteine (51) and BALB/c mice were immunized with the conjugate in Freund's adjuvant. 5 10 immune spleen cells were fused with 5 10 X63-Ag8.653 myeloma cells. Hybridoma clones were selected in HAT medium, and supernatants were screened against the Cx43 peptide by enzyme-linked immunosorbent assay. Two clones, F-3 (IgG) and D-7 (IgG), out of the 208 hybridomas screened were found to be reactive with the Cx43 peptide.

The immunoaffinity matrix was prepared with the D-7 monoclonal antibodies covalently bound to protein G-Sepharose 4B Fast Flow beads (Sigma). Briefly, mouse ascites fluid was subjected to a 50% ammonium sulfate precipitation (52) . The ammonium sulfate precipitate was resuspended in PBS and dialyzed overnight against PBS. The antibodies were then incubated with the protein G-Sepharose beads overnight at 4 °C with gentle end-over-end mixing. The beads were washed with 200 mM triethanolamine (TEA) in PBS (pH 8.0) and incubated for 2 h at room temperature with end-over-end mixing in 40 mM dimethylpalmilidate, 200 mM TEA, PBS (pH 8.0), followed by a 200 mM TEA (pH 8.0) wash and overnight incubation at room temperature in 40 mM dimethylpalmilidate, 200 mM TEA (pH 8.0) with end-over-end mixing. The beads were finally washed with 200 mM TEA (pH 8.0), incubated with 40 mM dimethylpalmilidate, 200 mM TEA (pH 8.0) for 2 h and incubated in 40 mM ethanolamine at room temperature for 2 h with end-over-end mixing. The beads were thoroughly washed with PBS and stored at 4°C in PBS containing merthiolate to prevent bacterial contamination (52, 53) .

Immunoaffinity Purification of Cx43 and pp60

Cx43 was purified from recombinant baculovirus-infected Sf-9 cells. At 72 h post-infection, 4 10 cells were harvested from a suspension culture and washed in PBS at 4 °C. The following steps were completed at 4 °C. Cells were disrupted with 75 strokes (pestle A) in a Dounce homogenizer in 5 ml of homogenization buffer (50 mM Tris-HCl (pH 7.2), 150 mM NaCl, 2 mM EDTA, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride). This homogenate was clarified in a Beckman TL100 ultracentrifuge at 100,000 g for 30 min at 4 °C. The supernatant was discarded and the pellet resuspended in 5 ml cell extraction buffer (10 mM Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100, 2 mM EDTA, 25 µg/ml leupeptin, 25 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride) by gentle passage through a 22-gauge, 1.5-inch needle. The resuspended sample was Dounce homogenized with 20 strokes (pestle A) and incubated on ice for 15 min. The sample was clarified in a Beckman TL100 ultracentrifuge at 400,000 g for 20 min at 4 °C. The supernatant was incubated with 1 ml of Cx43 monoclonal antibody-protein G-Sepharose for 3 h with end-over-end mixing at 4 °C. The slurry was then packed into a column and washed with cell extraction buffer, high salt wash buffer (10 mM Tris-HCl (pH 7.2), 1 M NaCl, 1% Triton X-100), high salt glycine wash buffer (100 mM glycine (pH 4.0), 1 M NaCl, 1% Triton X-100), and glycine wash buffer (100 mM glycine (pH 5.5), 1% Triton X-100). Cx43 was eluted from the immunoaffinity matrix with a low pH glycine buffer (100 mM glycine (pH 2.5), 1% Triton X-100), and the eluate was neutralized immediately with 1 M Tris-HCl (pH 8.0).

Activated pp60 was immunoaffinity-purified from Sf-9 cells infected with the recombinant baculovirus containing the c-src gene (Ref. 55; kindly provided by David Morgan). The immunoaffinity column was generated by covalently coupling the monoclonal antibody directed against pp60, mAb 327 (54) to protein A-agarose beads (Bio-Rad). The purification steps were performed as described previously (55) .

In Vitro Protein Kinase Assay

In vitro protein kinase assays were performed using immunoaffinity-purified, kinase-active pp60 and/or partially purified Cx43 in a reaction volume of 20 µl containing 0.10 µg of Cx43, 0.10 µg of pp60, kinase buffer (20 mM HEPES (pH 7.4), 12 mM MnCl, 20 mM MgCl, 1 mM dithiothreitol, 100 µM NaVO) and 10 µCi of [-P]ATP (DuPont NEN, NEG-002Z, 6000 Ci/mmol) for 15 min at room temperature. The reactions were stopped by the addition of 2 sample buffer and boiling.

For depletion experiments, the kinase reaction was performed in two incubations. The first incubation included partially purified Cx43, pp60, kinase buffer, and 5 nM unlabeled ATP. Following incubation for 15 min at room temperature, pp60 was immunodepleted from the reaction mixture by the addition of pp60 monoclonal antibodies (mAb 327) chemically coupled to protein A-agarose. The pp60 -depleted sample was then divided into two aliquots. To one portion, 10 µCi of [-P]ATP alone was added, and to the other portion, 10 µCi of [-P]ATP plus purified pp60 were added. These reactions were then incubated an additional 15 min at room temperature to permit radiolabeling to occur. The reactions were stopped with the addition of 2 sample buffer and boiling. The proteins were resolved on a 12% SDS-polyacrylamide gel. The gels were dried and autoradiographed using Kodak X-Omat XAR-5 film.

Generation and in Vitro Phosphorylation of a GST-Cx43CT Fusion Protein

A GST fusion protein containing the COOH tail region of Cx43 was generated by polymerase chain reaction (PCR) amplification (Perkin Elmer 480) of nucleotide sequences between 907-1356 of the Cx43 cDNA corresponding to amino acids 236-382 (GST-Cx43CT). The primer sequences for PCR amplification were 5`-GTATGGATCCGTTAAGGATCGCGTCAAGGG-3` and 5`-CTATGAATTCGCCGGTTTAAATCTCCAGGT-3`. The amplified DNA product was sequenced to confirm its authenticity. The PCR products were cloned into BamHI and EcoRI sites of the pGEX-KG expression vector (Ref. 56; generous gift from Jack Dixon). GST fusion proteins were expressed in Escherichia coli upon induction with 2 mM isopropyl--D-thiogalactopyranoside for 2 h at 37 °C. The cells were lysed by brief sonication in PBS and then incubated in 1% Triton X-100 for 15 min on ice. Insoluble material was removed by a 10 min spin on a microcentrifuge at 4 °C. The lysate was then incubated with glutathione-Sepharose 4B beads (Pharmacia) for 30 min followed by extensive washes with PBS.

For in vitro kinase assays, 15 µl of GST or GST-Cx43CT bound to glutathione-Sepharose 4B beads (washed once in kinase buffer) were incubated with purified kinase-active pp60, kinase buffer (20 mM HEPES (pH 7.4), 12 mM MnCl, 1 mM dithothreitol, 100 µM NaVO) and 10 µCi of [-P]ATP (DuPont NEN, NEG-002Z, 6000 Ci/mmol) in a total volume of 40 µl for 15 min at room temperature. Reactions were stopped with the addition of an equal volume of 2 sample buffer, boiled, and resolved on a 12% SDS-polyacrylamide gel.

Two-dimensional Tryptic Phosphopeptide Mapping

Tryptic phosphopeptide maps of phosphorylated Cx43 were prepared as described in Ref. 57. Briefly, v-src-transformed Rat-1 fibroblasts were metabolically labeled with 3 mCi of [P ] for 3 h at 37 °C and then solubilized in RIPA buffer. Cx43 was then immunoprecipitated from clarified lysates with CT368 peptide antiserum. For in vitro labeling experiments, partially purified Cx43 from infected Sf-9 cells was phosphorylated by activated pp60 in in vitro kinase reactions as described above. The radiolabeled proteins were resolved on 12% SDS-polyacrylamide gels and autoradiographed. Phosphorylated Cx43 was extracted from the wet, unfixed gel pieces and precipitated on ice with trichloroacetic acid. The precipitated Cx43 was then digested overnight with trypsin (Worthington, Freehold, NJ). The phosphotryptic peptides were resolved on cellulose thin-layer chromatography (TLC) plates (Curtin Matheson Scientific, Houston, TX) by electrophoresis in the first dimension using pH 1.9 buffer (2.5% (v/v) formic acid (88%), 7.8% (v/v) glacial acetic acid) for 1.25 h at 1000 V, followed by ascending chromatography in the second dimension using isobutyric acid buffer (62.5% (v/v) isobutyric acid, 1.9% (v/v) n-butanol, 4.8% (v/v) pyridine, 2.9% (v/v) glacial acetic acid). The positions of the phosphopeptides were determined by autoradiography using Kodak X-Omat XAR-5 film with the aid of an intensifying screen at -70 °C.


RESULTS

Expression of Cx43 in Sf-9 Cells

The time course of Cx43 production in recombinant baculovirus-infected Sf-9 cells was examined by harvesting cells at the specified times after infection. Cell lysates prepared from an equal number of cells were examined by Western blotting with antibody against the COOH-terminal peptide of Cx43. We observed that Cx43 was produced in the infected cells as early as 24 h post-infection at approximately 2.5-fold greater levels than detected in the Rat-1 fibroblast control (Fig. 1, A, lane3, and B). Expression of Cx43 was even more dramatic at 48 and 72 h post-infection with increases of 18- and 33-fold, respectively over Rat-1 fibroblast levels (Fig. 1, A, lanes5 and 7, and B). At these later times, multiple slower migrating forms of Cx43 were observed which are reminiscent of the phosphorylated Cx43 isoforms observed previously in mammalian cells (28, 38) . Uninfected Sf-9 cells did not express immunoreactive Cx43 (Fig. 1A, lanes2, 4, and 6).


Figure 1: Production and accumulation of Cx43 in recombinant baculovirus-infected Sf-9 insect cells. A, Western blotting of cell extracts from infected and uninfected Sf-9 cells harvested at the specified time points. Cell lysates from equal numbers of Rat-1 fibroblasts (lane1), uninfected Sf-9 cells (lanes2, 4, and 6), and infected Sf-9 cells (lanes 3, 5, and 7) were resolved on a 12% SDS-polyacrylamide gel and transferred to an Immobilon-P membrane. The samples were immunoblotted with Cx43 antibody and [I]-goat anti-rabbit IgG, as described under ``Materials and Methods.'' B, quantitation of Cx43 production in Sf-9 Cells. Immunoreactive protein bands were excised and quantitated with a Micromedics four-channel counter. The values were plotted as an increase of Cx43 in Sf-9 cells relative to the Cx43 level in Rat-1 fibroblasts.



Subcellular Localization of Cx43

Endogenously expressed Cx43, like other members of the gap junction protein family, localize in the plasma membranes of mammalian cells or Xenopus oocytes microinjected with connexin mRNA (11, 41) . To determine if Cx43 was also localized to the plasma membrane in recombinant baculovirus-infected Sf-9 cells, biochemical subcellular fractionation of infected and uninfected Sf-9 cells was performed. Cytosolic and particulate proteins were separated by SDS-polyacrylamide gel electrophoresis and Cx43 was detected by Western blot analysis. The particulate fraction of infected Sf-9 cells contained Cx43 with apparent molecular weights ranging from approximately 43,000 to 47,000 (Fig. 2A, lane4). Cx43 was nearly undetectable in the soluble, cytosolic fraction (Fig. 2A, lane3), and it was not detected in either the particulate or cytosolic fractions of uninfected Sf-9 cells (Fig. 2A, lanes1 and 2).


Figure 2: Localization of Cx43 in Sf-9 cells by biochemical subcellular fractionation and immunofluorescence microscopy. A, cytosolic (lanes1 and 3) and particulate (lanes2 and 4) proteins of uninfected (lanes1 and 2) and infected (lanes3 and 4) Sf-9 cells 72 h post infection were resolved on a 12% SDS-polyacrylamide gel, transferred to an Immobilon-P membrane, and reacted with Cx43 antibody, as described under ``Materials and Methods.'' B, uninfected (panels1 and 2) and infected (panels3 and 4) Sf-9 cells 48 h post-infection were fixed, permeabilized, and then examined by indirect immunofluorescent staining with Cx43 antibody and fluorescein isothiocyanate-conjugated goat anti-rabbit antibody. Phase (panels 1 and 3) and fluorescent (panels2 and 4) images were photographed with a Zeiss Axioplane Universal microscope with epifluorescence.



To confirm these biochemical results and to identify the specific cellular membranes containing Cx43, immunofluorescence microscopy studies were conducted on Cx43 baculovirus-infected Sf-9 cells. Triton X-100-permeabilized Sf-9 cells were incubated with Cx43 COOH-terminal peptide antibody, and the immune complexes were detected with fluorescein-conjugated secondary antibody. Infected cells demonstrated a strong Cx43 signal in the cell periphery with only a faint signal in the cytoplasm (Fig. 2B, panel4). These results are consistent with the notion that Cx43 is localized predominantly to the plasma membranes of infected Sf-9 cells. Specificity of the Cx43 antibody reaction was demonstrated by the lack of a specific response in uninfected Sf-9 cells (Fig. 2B, panel2).

Phosphorylation of Cx43 in Sf-9 Cells

The postranslational modification of Cx43 by serine phosphorylation in various cells is associated with the modulation of channel gating activity and its membrane localization (35, 38, 58, 59) . Cx43 immunoprecipitated from fibroblasts migrates on SDS-polyacrylamide gels predominantly as the nonphosphorylated 43-kDa form and two slower migrating bands of 45 and 47 kDa, each of which represents serine phosphorylated isoforms of Cx43 (28, 38, 39).

To determine if Cx43, expressed in recombinant baculovirus-infected Sf-9 cells, was also phosphorylated, S- and P-labeled Cx43 were immunoprecipitated from uninfected and infected Sf-9 cells and Rat-1 fibroblasts and resolved by SDS-polyacrylamide gel electrophoresis. Five major forms of Cx43 with apparent molecular mass values of 43, 44, 45, 47, and >47 kDa were isolated from S-labeled infected, but not uninfected, Sf-9 cells (Fig. 3A, lanes4 and 6). Immunoprecipitation of P-labeled Cx43 from infected Sf-9 cells demonstrated that the slower migrating forms of Cx43 were phosphorylated (Fig. 3B, lane 4). Four phosphoproteins with corresponding molecular mass values of 44, 45, 47, and >47 kDa were detected, indicating that the Cx43 produced in the baculovirus expression system is phosphorylated in a manner similar to that observed in Rat-1 fibroblasts. However, there were differences in the extent of phosphorylation of the Cx43 isoforms isolated from the two cell types (Fig. 3B, compare lanes2 and 4). The predominant phosphorylated Cx43 in Rat-1 fibroblasts was the 45-kDa protein, whereas the 47-kDa form of Cx43 predominated in infected Sf-9 cells. Also, the >47-kDa band was readily detected in infected Sf-9 cells, but to a lesser extent in the Rat-1 fibroblasts. Phosphoamino acid analysis showed that Cx43 expressed in infected Sf-9 cells and Rat-1 fibroblasts contained only Ser(P) (Fig. 3C).


Figure 3: Phosphorylation of Cx43 expressed in recombinant baculovirus-infected Sf-9 insect cells. Rat-1 fibroblasts, uninfected (U), and infected (I) Sf-9 cells were labeled with either EXPRESS (panelA) or [P] (panelB). Cx43 was immunoprecipitated from cell lysates with either nonimmune rabbit serum (odd numbered lanes) or antiserum against Cx43 (even numbered lanes) and resolved on 7.5-15% SDS-polyacrylamide gel as described under ``Materials and Methods.'' C, phosphoamino acid analysis of [P]Cx43 from the Rat-1 fibroblasts and infected Sf-9 cells. The positions of ninhydrin-stained unlabeled Ser(P) (P-S), Thr(P) (P-T), and Tyr(P) (P-Y) are outlined. The directions of migration for the first dimension (pH 1.9) and second dimension (pH 3.5) on thin-layer cellulose plates are indicated by the arrows. All autoradiograms were prepared on Kodak X-Omat XAR-5 film at -70 °C.



Immunoaffinity Purification of Cx43

For in vitro phosphorylation studies, rapid partial purification of Cx43 was achieved by immunoaffinity chromatography using a monoclonal antibody against a Cx43 peptide (residues 241-254, clone D-7) covalently coupled to protein G-Sepharose. The solubilized, particulate fraction prepared from 72 h post-infected Sf-9 cells was incubated with the immunoaffinity matrix (Fig. 4, lane1). Following extensive washing to remove nonspecific proteins, Cx43 was eluted from the matrix with a low pH buffer. Coomassie Blue staining showed that the predominant protein isolated was the nonphosphorylated 43-kDa form of Cx43 (Fig. 4, lanes 4 and 5). The phosphorylated Cx43 isoforms were also present to a lesser extent in the purified preparations. The identity of this immunoaffinity product as Cx43 was confirmed by Western blotting utilizing a polyclonal antibody directed against COOH-terminal peptide of Cx43 (data not shown). Trace levels of non-Cx43 proteins were detected at 70 and 31 kDa by Coomassie staining. The identities of these proteins are unclear at the present time.


Figure 4: Immunoaffinity purification of Cx43 from recombinant baculovirus-infected Sf-9 cells. Cx43 was purified from recombinant baculovirus-infected Sf-9 cells using a Cx43 monoclonal antibody chemically coupled to protein G-Sepharose, as described under ``Materials and Methods.'' After extensive washing, Cx43 was eluted from the column with a low pH buffer. Samples were resolved on a 12% SDS-polyacrylamide gel and stained with Coomassie Blue. Lanes1 and 2 represent the loading sample (LS) and flow-through (FT), respectively. Lanes 4-11 represent elution fractions 1-8 (E). Cx43 was eluted predominantly in fractions 2 and 3 (lanes4 and 5). The sizes of the molecular size markers are as indicated at the leftmargin.



pp60 -mediated Phosphorylation of Cx43 in Vitro

Previous studies have demonstrated that Cx43 is phosphorylated on tyrosine in mammalian cells transformed by pp60(38, 39, 40) and in Xenopus oocytes microinjected with both Cx43 and pp60 mRNAs (41) . These studies suggested that the tyrosine kinase responsible for phosphorylating Cx43 was pp60. The availability of these purified proteins permitted us to examine the ability of pp60 to directly phosphorylate Cx43 in in vitro kinase assays. Several phosphorylated proteins were observed in these kinase reactions. One major phosphorylated protein of approximately 44-47 kDa comigrated with the Coomassie-stained, purified Cx43 (Fig. 5, compare panelA, lane3 to panelB, lane4). The second major phosphorylated protein migrated around 60 kDa and represents autophosphorylated pp60, since it comigrated with the major phosphorylated product present in reactions consisting only of pp60 (Fig. 5A, lanes1 and 3). Phosphoamino acid analyses of these in vitro phosphorylated products revealed only Tyr(P) (Fig. 5C). In similar experiments, a baculovirus-expressed pp60, purified independently under different conditions (Ref. 60; generously provided by Martin Broome, Salk Institute), also phosphorylated Cx43 on tyrosine (data not shown). To determine if our Cx43 preparation contained endogenous kinases capable of phosphorylating Cx43, a kinase reaction was performed in the absence of pp60. No phosphorylated proteins were observed in this control reaction (Fig. 5A, lane2). These data are consistent with the idea that activated pp60 phosphorylates Cx43 directly in vitro.


Figure 5: Phosphorylation of Cx43 in pp60 kinase reactions. pp60 and Cx43 were immunoaffinity-purified as specified under ``Materials and Methods.'' A, results from the in vitro kinase assay with pp60 alone (lane1), Cx43 alone (lane2), and pp60 together with Cx43 (lane3). B, Coomassie-stained SDS-polyacrylamide gel of the in vitro kinase reactions shown in panelB. Cx43 is indicated by the brackets. The samples were resolved on a 7.5-15% SDS-polyacrylamide gel. The gel was dried and autoradiographed at -70 °C. The sizes of the molecular size standards are indicated between panelsA and B. Positions of phosphorylated pp60 and Cx43 are shown at the left. C, phosphoamino acid analysis of [P]pp60 and Cx43 from the in vitro kinase reaction. The positions of ninhydrin-stained, unlabeled Ser(P) (P-S), Thr(P) (P-T), and Tyr(P) (P-Y) are outlined. The directions of migration for the first dimension (pH 1.9) and second dimension (pH 3.5) on thin-layer cellulose plates are indicated by the arrows.



To rule out the possibility that Cx43 was phosphorylated by an activated endogenous tyrosine kinase that was present in the partially purified Cx43 preparation and activated by pp60 phosphorylation, we conducted a two-stage kinase reaction in which pp60 was initially present, but then immunodepleted to reveal the activity of any activated endogenous tyrosine kinase(s). In this experiment, a complete kinase reaction, containing activated pp60 and Cx43, was first performed with unlabeled ATP (5 nM) to permit pp60 to phosphorylate and possibly activate any putative tyrosine kinase(s) present in the reaction mix. pp60 was then immunodepleted from this reaction mix by incubation with a pp60 monoclonal antibody conjugated to protein A-agarose beads, and the kinase reaction was continued in the presence of 10 µCi of [-P]ATP to detect the activity of any putative, activated kinases. Under these experimental conditions, it was expected that any putative pp60 -activated tyrosine kinase might reveal itself in the second kinase reaction by phosphorylating Cx43 in the absence of pp60. However, our results demonstrated that phosphorylation of Cx43 did not occur in pp60 immunodepleted reactions (Fig. 6, lane2). The absence of autophosphorylated pp60 in this reaction indicated that the immunodepletion of pp60 was effective (Fig. 6, lane2). Furthermore, when pp60 was added back to the depleted reaction, Cx43 phosphorylation and pp60 autophosphorylation were observed, which indicated that Cx43 was not altered or removed during the immunodepletion step and that the ability of the depleted reaction mix to support protein phosphorylation was not compromised (Fig. 6, lane3). Thus, these results demonstrated that the partially purified Cx43 preparation did not contain any detectable endogenous kinase(s) capable of phosphorylating Cx43. Control kinase reactions, performed in the presence of 5 nM unlabeled ATP with 10 µCi of [-P]ATP, supported phosphorylation of Cx43 and pp60 (Fig. 6, lane1), which suggested that pp60 should have been capable of phosphorylating a putative tyrosine kinase present in the kinase reaction mix. The lower levels of radiolabeled Cx43 and pp60 observed in this reaction, compared to reactions containing 10 µCi of [-P]ATP alone (see Fig. 5A, lane3), was probably because of dilution of the radiolabel by the unlabeled ATP.


Figure 6: Phosphorylation of Cx43 in kinase reactions depleted of pp60. Control kinase reaction performed with pp60 and Cx43 in the presence of 10 µCi of [-P]ATP and 5 nM ATP (lane1). Kinase reaction performed first with pp60 and Cx43 in the presence of 5 nM ATP; pp60 was then immunodepleted, and the reaction was continued with 10 µCi of [-P]ATP (lane2). Kinase reaction performed as described for lane2 but pp60 was added back to the depleted reaction (lane3). Samples were resolved on a 10% SDS-polyacrylamide gel. The gel was dried and autoradiographed at -70 °C. The positions of pp60src and Cx43 are indicated at the leftmargin.



A Western blot of the purified pp60 and Cx43 samples, performed with polyclonal antibodies (BC3) against pp125(42) , was negative, which excluded the possibility that the tyrosine kinase pp125 was associated with either purified pp60 or Cx43 preparations (data not shown).

Phosphotryptic Peptide Mapping of Cx43 Phosphorylated in Vivo and in in Vitro Kinase Reactions

Two-dimensional tryptic phosphopeptide analysis was performed to determine if the sites of Cx43 phosphorylation mediated by pp60in vitro corresponded with sites of tyrosine phosphorylation on Cx43 from in vivo labeled v-src-transformed fibroblasts. Four phosphopeptides (labeled 1-4) of full-length Cx43, purified from Sf-9 cells and phosphorylated in vitro by pp60, migrated similarly to phosphopeptides from Cx43 radiolabeled with [P ] in v-src-transformed cells (Fig. 7, panelsA and B). Analysis of a mixture of these two samples confirmed that the four phosphopeptides labeled in vitro comigrated with phosphopeptides from the in vivo labeled sample (Fig. 7, panelC). Peptides 1 and 4 of Cx43 from v-src-transformed cells correspond to phosphotyrosine-containing peptides a and c previously reported in Ref. 61. Phosphoamino acid analyses of peptides 2 and 3 revealed that they also contained Tyr(P).()


Figure 7: Two-dimensional tryptic phosphopeptide maps of P-labeled Cx43. Cx43 from radiolabeled v-src-transformed fibroblasts was immunoprecipitated with Cx43 CT368 peptide antiserum and resolved on a SDS-polyacrylamide gel as described under ``Materials and Methods.'' Partially purified, full-length Cx43 from infected Sf-9 cells and the GST carboxyl tail Cx43 fusion protein were phosphorylated in vitro in the presence of pp60 as described under ``Materials and Methods.'' Figure shows phosphotryptic peptides of: A, full-length Cx43 phosphorylated in vitro; B, Cx43 from v-src-transformed fibroblasts; C, mixture of in vivo labeled Cx43 and full-length Cx43 phosphorylated in vitro; D, GST-Cx43CT phosphorylated in vitro; E, mixture of GST-Cx43CT phosphorylated in vitro and full-length Cx43 phosphorylated in vitro. Origins are indicated by the arrowheads. The directions of migration are indicated by the arrows. The inset in panelD shows the phosphoamino acid analysis results of the phosphorylated GST-Cx43CT fusion protein. The positions of the unlabeled Ser(P) (P-S), Thr(P) (P-T), and Tyr(P) (P-Y) standards are outlined.



A bacterially expressed GST-fusion protein containing the carboxyl tail region (amino acids 236-382) of Cx43 (GST-Cx43CT) was generated to determine if the putative pp60 -mediated phosphorylation of Cx43 occurred in this domain. The bacterially expressed GST-Cx43CT protein was purified by absorption on glutathione-Sepharose. The GST-Cx43CT fusion protein bound to glutathione-Sepharose was phosphorylated only on tyrosine by activated pp60 (Fig. 7, panelD, inset). Control GST protein bound to Sepharose beads was not phosphorylated under identical conditions (data not shown). The tryptic peptide map of phosphorylated GST-Cx43CT had a similar pattern consisting of the same four phosphopeptides observed in the in vitro phosphorylated whole Cx43 isolated from Sf-9 cells (Fig. 7, panelD). A mixture of the tryptic digests of the two samples demonstrated the comigration of the four phosphopeptides (Fig. 7, panelE). However, the intensities of the phosphorylated peptides differed between the two samples. Peptide 4 represented the major phosphotryptic spot in the GST-Cx43CT protein, whereas peptides 1 and 2 were the major phosphopeptides observed in in vitro phosphorylated full-length Cx43 isolated from the Sf-9 cells (Fig. 7, compare panelsA and D). Taken together, these results suggested that pp60 -mediated phosphorylation of Cx43 in vitro mimics similar phosphorylation events occurring in v-src-transformed fibroblasts and that the tyrosine phosphorylation of Cx43 may be occurring primarily in the carboxyl tail region of the molecule.

Phosphorylation of Cx43 in Sf-9 Cells Coinfected with pp60 and Cx43 Recombinant Baculoviruses

Several studies have utilized Sf-9 cells coinfected with recombinant baculoviruses to demonstrate protein kinase-substrate interactions (62, 63) . We also examined the ability of pp60 to phosphorylate Cx43 in coinfected Sf-9 cells. Phosphorylated forms of both pp60 and Cx43 were immunoprecipitated from these cells (Fig. 8A, lanes8 and 9). Cx43 from the coinfected cells was phosphorylated primarily on Ser(P), but also contained levels of Tyr(P) (Fig. 8B) that were comparable to the phosphorylated Cx43 expressed in v-src-transformed Rat-1 fibroblasts (Fig. 8, panelA, lane3 and panelB). In contrast, Cx43 expressed in Sf-9 cells infected with the Cx43 baculovirus alone was phosphorylated only on serine residues as described above (Fig. 8, panelA, lane6 and panelB). Thus, these coinfection experiments further support the conclusion that a direct kinase-substrate interaction between pp60 and Cx43 exists.


Figure 8: Phosphorylation of Cx43 in insect Sf-9 cells co-expressing pp60. A, Rat-1 v-src-transformed fibroblasts, monoinfected (Cx43 recombinant baculovirus), and coinfected (Cx43 and pp60 recombinant baculoviruses) Sf-9 cells were labeled with [P]. Cell lysates were immunoprecipitated with either nonimmune rabbit serum (lanes1, 4, and 7), mAb 327 monoclonal antibody specific for pp60 (lanes2, 5, and 8), or antiserum directed against Cx43 (lanes3, 6, and 9). Immunoprecipitates were resolved on a 7.5-15% SDS-polyacrylamide gel and autoradiographed. The positions of pp60 and Cx43 are shown at the left. Molecular size standards are shown at the right. B, phosphoamino acid analysis of [P]Cx43. The positions of ninhydrin-stained, unlabeled Ser(P) (P-S), Thr(P) (P-T), and Tyr(P) (P-Y) are outlined. The directions of migration of the first dimension (pH 1.9) and second dimension (pH 3.5) are indicated by the arrows.




DISCUSSION

Our laboratory and others have previously demonstrated that Cx43 is phosphorylated on tyrosine in Rous sarcoma virus-transformed fibroblasts (38, 40) . This phosphorylation event is tightly associated with the kinase activity and membrane localization of pp60. In cells expressing temperature-sensitive pp60, phosphorylation of Cx43 on tyrosine correlated closely with activation of the pp60 kinase activity and the reduction of GJC (39) . Moreover, tyrosine phosphorylation of Cx43 and disruption of its function were not observed in cells containing kinase-active but nonmyristylated pp60, which does not localize to plasma membranes (39, 40) . In this study, we present data which strongly suggest that pp60 is one of the tyrosine kinases responsible for directly phosphorylating Cx43 in src-transformed fibroblasts. In support of this conclusion, we have demonstrated that purified, activated pp60 phosphorylates Cx43 in in vitro kinase reactions and that the tryptic phosphopeptides of in vitro phosphorylated Cx43 represent a subset of those obtained from Cx43 metabolically labeled in v-src-transformed cells.

We chose the baculovirus eucaryotic expression system for the production of Cx43 because it has been used successfully to express high levels of native, post-translationally modified protein (55, 64) . Cx43 was expressed in the recombinant baculovirus-infected Sf-9 cells at levels 33 times greater than those observed with Rat-1 fibroblasts (Fig. 1). Biochemical subcellular fractionation and immunofluorescence microscopy demonstrated that Cx43 was found in the plasma membranes of the Sf-9 cells (Fig. 2). We also observed that the expressed Cx43 was postranslationally modified by endogenous serine kinases in insect cells (Fig. 3).

Baculovirus-expressed Cx43, partially purified with a monoclonal antibody immunoaffinity matrix, was used in in vitro kinase assays with immunoaffinity-purified, activated pp60 to demonstrate that Cx43 is phosphorylated on tyrosine in the presence of activated pp60 (Fig. 5). Several control reactions assured us that the tyrosine kinase responsible for phosphorylating Cx43 in vitro was indeed pp60. First, kinase reactions performed with the partially purified preparation of Cx43 alone did not result in the phosphorylation of Cx43 (Fig. 5B). Second, because it was possible that another tyrosine kinase, which required activation by pp60, was present in either the pp60 or Cx43 preparations, we performed a two-stage kinase reaction in which the putative tyrosine kinase was first activated by pp60. Its activity was then measured in the second stage in the absence of pp60. This experimental protocol failed to produce in vitro phosphorylated Cx43. However, Cx43 from the pp60 -depleted reaction mixture could be phosphorylated when pp60 was added back (Fig. 6). Third, p125, which has been reported to be associated with pp60(44) , was not detected in the pp60 or Cx43 preparations by immunoblotting. Therefore, these results suggest that Cx43 is phosphorylated directly by activated pp60 in these in vitro assays.

This conclusion was reinforced by our demonstration of pp60 's ability to directly phosphorylate a GST-Cx43CT fusion protein expressed in bacteria. This substrate, purified from bacterial cell lysates on glutathione-Sepharose, is unlikely to contain endogenous tyrosine kinases capable of phosphorylating Cx43 in vitro.

To examine whether the ability of activated pp60 to phosphorylate Cx43 in vitro reflected its ability to do so in intact v-src-transformed cells, we compared the phosphotryptic peptides of in vitro and in vivo phosphorylated Cx43 by two-dimensional peptide mapping. These experiments demonstrated that four of the Cx43 peptides phosphorylated in vitro represented a subset of those obtained from Cx43 phosphorylated in v-src-transformed fibroblasts (peptides 1-4, Fig. 7 ). The corresponding four phosphopeptides of in vivo labeled Cx43 contained phosphotyrosine (Ref. 61, and unpublished observations). Peptides 1 and 4 (Fig. 7) were identified in a previous study characterizing the phosphorylation of Cx43 in v-src-transformed cells (labeled as peptides a and c in Ref. 61). Peptides 2 and 3 (Fig. 7) were also detected in this previous study, but at a reduced level of phosphorylation (61) . The reasons for the differences in phosphorylation levels of peptides 2 and 3 are not clear at this time.

We also examined the phosphorylation of Cx43 in Sf-9 cells coinfected with the Cx43 and pp60 baculoviruses. Phosphoamino acid analysis demonstrated that the Cx43 expressed in these coinfected cells contained both Tyr(P) and Ser(P). In contrast, Cx43 expressed in Sf-9 cells infected with only the Cx43 recombinant baculovirus contained only Ser(P). Our attempts to measure an alteration of the gating activity of Cx43 in Sf-9 cells, expressed in the presence or absence of pp60, were unsuccessful due to the decreased adhesiveness of the cells following baculovirus infection.

Taken together, these data demonstrated that Cx43 is phosphorylated directly by activated pp60 in in vitro kinase reactions and support the hypothesis that Cx43 is phosphorylated by pp60 in intact v-src-transformed fibroblasts. However, because of the existence of enzymes like pp125, which is associated with pp60, we cannot exclude the possibility that Cx43 may also be phosphorylated in vivo by other tyrosine kinases besides pp60.

The apparent ability of pp60 to phosphorylate four phosphotryptic peptides of full-length Cx43 suggested the possibility of multiple Tyr(P) sites in Cx43. These results differ from those published previously, which indicated that phosphorylation of tyrosine 265 in Cx43 expressed in Xenopus oocytes was sufficient to disrupt junctional conductance (41) . Because these multiple phosphopeptides may possibly result from events such as incomplete tryptic cleavage or differential serine phosphorylation, we are identifying the Tyr(P) sites present in these four Cx43 tryptic peptides. The same phosphopeptides also resulted from pp60 's phosphorylation of the GST-Cx43CT fusion protein (Fig. 7), which contains the COOH-terminal cytoplasmic region of Cx43. Therefore, the potential phosphorylation sites of interest must be located between amino acids 236-382 of Cx43. Thus, one of the four phosphopeptides from Cx43, phosphorylated in v-src-transformed fibroblasts, may also contain phosphorylated tyrosine 265. Site-directed mutagenesis studies should help to clarify the relative importance of these potentially different Cx43 phosphorylation sites in pp60 's ability to interrupt GJC in mammalian cells.

An increased serine phosphorylation on Cx43 has also been observed in v-src-transformed cells (40, 61) . v-src has been demonstrated to activate protein kinase C and thus may induce serine phosphorylation of Cx43 by this mechanism (65) . pp60 also associates with Shc (66, 67) and activates the ras-raf signaling pathway which may result in mitogen-activated protein kinase-mediated phosphorylation of Cx43 (68) . Additional v-src-dependent serine kinases may also be involved but are yet to be identified. Serine phosphorylation of Cx43 induced by the ras oncogene or epidermal growth factor is correlated with down-regulation of GJC (15, 28, 35) . Epidermal growth factor's effects on Cx43 phosphorylation are independent of PKC, but may involve activated mitogen-activated protein kinase (35) . The function of the observed increase in serine phosphorylation in v-src-transformed cells is at present unknown. It is possible that a combination of serine and tyrosine phosphorylation of Cx43 contribute to the regulation of the gap junction channel.

Multiple pp60 substrates have been identified in src-transformed cells, including cytoskeletal proteins (69-71), the epidermal growth factor receptor (72) , focal adhesion kinase (p125) (42, 43) , and an RNA-binding protein (73, 74) . The identification of pp60 substrates is critical to the understanding of the mechanisms involved in pp60 's effects on growth and cellular transformation. It is possible that the phosphorylation of only one or two substrates may be sufficient for cellular transformation. Alternatively, multiple pp60 substrates may work in concert to initiate and maintain the different properties associated with altered cell growth and behavior. Based on previous studies and data presented here, we propose that Cx43 also serves as a direct substrate of pp60. Several studies have suggested that the maintenance of intercellular GJC is one parameter critically involved in normal growth control (6, 75) . pp60 -mediated tyrosine phosphorylation of Cx43 and the associated disruption of GJC could be one mechanism by which pp60 mediates some aspects of cellular transformation and loss of growth control.


FOOTNOTES

*
This research was supported by Grant CA 52098 from the NCI, National Institutes of Health and Grant VM-21A from the American Cancer Society (to A. F. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Molecular Biology and Virology Laboratory, Salk Institute, La Jolla, CA 92138.

To whom correspondence should be addressed. E-mail: aflau@uhunix.uhcc.hawaii.edu.

The abbreviations used are: GJC, gap junctional communication; Cx43, connexin 43; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; MES, 2-(N-morpholino)ethanesulfonic acid; mAb, monoclonal antibody; TEA, triethanolamine; RSV, Rous sarcoma virus.

W. Kurata, unpublished observations.


ACKNOWLEDGEMENTS

We thank E. Beyer for Cx43 cDNA, J. Brugge for mAb 327, D. Morgan and R. Erikson for pp60 recombinant baculoviruses, M. Broome for purified, kinase-active pp60, J. T. Parsons for antibodies against pp125, and J. Dixon for pGEX clones. We also thank K. Martyn for suggesting the depletion experiments, W. Kurata for technical support, K. Martyn and B. Warn-Cramer for critical review of manuscript, and L. Kuriyama for assistance in the preparation of the manuscript.


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