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Previous Article | Next Article 
Molecular and Cellular Biology, May 2004, p. 4351-4360, Vol. 24, No. 10
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.10.4351-4360.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Phosphorylation of Specific Serines in the Connecting Segment of the ß4 Integrin Regulates the Dynamics of Type II Hemidesmosomes
Division of Cancer Biology and Angiogenesis, Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02115,
Received 9 October 2003/ Returned for modification 2 December 2003/ Accepted 18 February 2004
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ABSTRACT |
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6ß4 is an essential component of the hemidesmosome and a target of such regulation. Epidermal growth factor (EGF) can induce hemidesmosome disassembly by a mechanism that involves serine phosphorylation of the ß4 integrin subunit. Using a combination of biochemical and mutational analyses, we demonstrate that EGF induces the phosphorylation of three specific serine residues (S1356, S1360, and S1364) located within the connecting segment of the ß4 subunit and that phosphorylation on these residues accounts for the bulk of ß4 phosphorylation stimulated by EGF. Importantly, phosphorylation of these serines is critical for the ability of EGF to disrupt hemidesmosomes. Using COS-7 cells, which assemble hemidesmosomes type II upon exogenous expression of the 6ß4 integrin, we observed that expression of a ß4 construct containing Ser
Ala mutations of S1356, S1360, and S1364 reduced the ability of EGF to disrupt hemidesmosomes and that this effect appears to involve cooperation among these phosphorylation sites. Moreover, expression of SerAsp mutants that mimic constitutive phosphorylation reduced hemidesmosome formation. Protein kinase C- (PKC-) is the kinase responsible for phosphorylating at least two of these serines, based on in vitro kinase assays, peptide mapping, and mutational analysis. Together, these results highlight the importance of serine phosphorylation in regulating type II hemidesmosome disassembly, implicate a cluster of serine residues within the connecting segment of ß4, and argue for a key role for PKC- in regulating these structures. |
INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the most important molecules mediating stable cell attachment to the basal lamina is the integrin 6ß4, a laminin receptor that links the basal lamina to the intermediate filament network and that is a main component of a multiprotein structure known as the hemidesmosome (4, 14, 24). This integrin interacts with several hemidesmosomal proteins directly or indirectly, namely HD1/plectin, bullous pemphigoid antigen 1 (BPAG1), and BPAG2, and it is through the first two of these proteins that the ß4 subunit is linked indirectly to the cytokeratins (4, 14). More specifically, the ß4 subunit contains several structural and functional regions within its unique 1,000-amino-acid-long cytoplasmic tail that are important for its association with these hemidesmosomal proteins (16, 31, 42). These regions include two pairs of FN type III repeats separated by a connecting segment. The ß4 subunit has at least two areas where it binds to plectin: one encompassing part of the FN type III repeat 2 plus a region of the connecting segment and another one encompassing the last FN repeat plus the carboxy-terminal residues (27, 28, 30, 36). The FN type III repeats 3 and 4 are also important for binding BPAG1 and -2 (3, 18). Two types of hemidesmosomes have been described. Type I hemidesmosomes are present in skin and several types of epithelia, and they are composed of 6ß4, HD1/plectin, BPAG1, and BPAG2 (4, 14). Type II hemidesmosomes are present in intestinal epithelia and they contain only 6ß4 and HD1/plectin (11, 43).
The 6ß4 integrin is critical for the formation of the hemidesmosome, and the loss of its expression in certain genetic diseases or in ß4 null mice results in the disappearance of these structures, producing an epithelium that is mechanically and functionally deficient (8, 29, 44). In the absence of ß4 mutations, hemidesmosome disassembly also occurs during wound healing and carcinoma invasion and it facilitates cell migration, even though cells can still express all of the hemidesmosomal proteins (13, 19, 35). Growth factors such as epidermal growth factor (EGF) have been used to induce hemidesmosome disassembly and to study the mechanisms involved (7, 21, 35). EGF stimulates phosphorylation of the ß4 subunit on both serine and tyrosine residues (21, 35). Phosphorylated serine residues, which proportionally constitute the vast majority of the phosphorylated residues in the ß4 subunit, are dependent on a functional protein kinase C (PKC) pathway, and the inhibition of conventional PKCs results in the inhibition of both hemidesmosome disassembly and ß4 phosphorylation (35). None of these serine residues have been identified. In this study, we identify three phosphoserine residues located in the connecting segment of the ß4 subunit that constitute approximately 50% of total phosphorylation of ß4 that is induced by EGF. We show that at least two of these serines are phosphorylated by PKC-, an isoform that has been previously shown to play a major role in hemidesmosome dynamics (35). Site-specific mutations of these sites that mimic constitutive phosphorylation reduce the formation of type II hemidesmosomes. In contrast, mutations that nullify phosphorylation of these serines reduce the ability of EGF to disrupt hemidesmosomes.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following antibodies were used in this study: rat GoH3 monoclonal antibody (MAb [integrin 6 specific]) from Chemicon and rat MAb 439-9B (integrin ß4 specific) (9) provided by Rita Falcioni (Regina Elena Cancer Institute, Rome, Italy). A peptide-specific antiserum elicited against the last 20 amino acids of the COOH terminus of the ß4 subunit was prepared commercially. The HD1/plectin MAb was obtained from Becton-Dickinson (San Diego, Calif.). Tetramethyl rhodamine isothiocyanate- and Cy2-conjugated secondary antibodies were purchased from Jackson Immunolabs.
Laminin-1, prepared from the EHS sarcoma, was provided by Hynda Kleinman (National Institute of Dental Research, Bethesda, Md.). Collagen type I was purchased from Collagen Corp. (Palo Alto, Calif.). Human recombinant EGF, -chymotrypsin, Asp-N-endoproteinase, and V8 protease were purchased from Sigma Chemical (St. Louis, Mo.). Purified PKC isoforms were purchased from Calbiochem (San Diego, Calif.). Gö6976 was from Alexis Biochemical.
Metabolic radiolabeling. Cells were plated on laminin-1-coated dishes for 2 h and then washed several times with phosphate-free Dulbecco's modified Eagle's medium and starved for 1 h before adding 32PO4 (1.0 mCi/ml; NEN, Boston, Mass.) and incubating for 3 more h. The cells were then stimulated with EGF (100 ng/ml) for 15 min, washed several times with phosphate-buffered saline (PBS) and extracted in radioimmunoprecipitation assay buffer containing 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 0.5% deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 10-µg/ml each leupeptin, pepstatin A, and aprotinin, and 50 mM Tris-HCl (pH 7.5). The samples were immunoprecipitated using the 439-9B antibody, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Micropore).
In vitro PKC kinase assay.
After immunoprecipitation of 6ß4 with the 439-9b antibody and antirat agarose in radioimmunoprecipitation assay buffer and washing several times with kinase buffer (50 mM Tris/HCl [pH 7.5], 10 mM MgCl2), the beads were resuspended in 50 µl of kinase buffer containing 1 µg of a PKC isoform (PKC-, -ßI, -ßII, and -
from Calbiochem), 50 µM ATP, 140 µM phosphatidylserine, 4 µM 1,2-dioleoyl-SN-glycerol (DOG; Avanti), and 10 µCi of [
-32]ATP (NEN). The reaction was carried out for 20 min at 30°C. The samples were centrifuged, washed, and eluted in sample buffer before PAGE and electrotransfer onto a PVDF membrane.
Analysis of the radiolabeled ß4 integrin subunit.
The PVDF membranes containing the ß4 immunoprecipitates were exposed to a phosphor screen (Bio-Rad), and the area of the membrane that contained the ß4 subunit was excised with a razor. For phosphoamino acid analysis, the excised band (or eluted phosphopeptide [see below]) was acid hydrolyzed and resolved using two-dimensional thin-layer electrophoresis (TLE) and exposed to a phosphor screen following standard techniques (25). For phosphopeptide mapping, the band was digested with trypsin as described previously (25) and run on a thin-layer chromatography (TLC) plate (cellulose) using a pH 1.9 buffer during the electrophoresis separation in the first dimension (TLE) and then a standard chromatography buffer in the second dimension (TLC), followed by exposure of the plate to a phosphor screen. The phosphopeptide profile of ß4 was compared with a prediction profile generated by PhosphoPepsort4 (www.genestream.org) to identify candidate sequences. The cellulose area corresponding to the spots of interest was scraped off, and the phosphopeptides were eluted for further analysis. Manual Edman degradation was performed using standard techniques (25). The eluted phosphopeptides were subject to 2 to 5 cycles of degradation and then resolved using TLE and exposed to a phosphor screen. Diagnostic secondary digestions from the phosphopeptides were performed as described previously (25) by digesting the eluted phosphopeptides with either -chymotrypsin (1 µg/ml in ammonium bicarbonate buffer at pH 8 [see above]), V8 protease, or Asp-N-endoproteinase (1 µg/ml, pH 7.6). The digests were inactivated and resolved using TLE alongside undigested controls.
Site-directed mutagenesis and cDNA transfections.
Single or multiple amino acid substitutions on human wild-type ß4 cDNA were made by generating PCR fragments into which the desired mutations had been introduced by means of appropriately designed primers following standard techniques (6). The resulting point mutations were confirmed by dideoxy sequencing. The vectors (pcDNA4 plasmid; InVitrogen) containing the mutant ß4 cDNAs (1 µg) were cotransfected with an 6 cDNA (1 µg) (39) into the COS-7 cells by using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Myristylated PKC- (35) was kindly provided by Alex Toker (Beth Israel Deaconess Medical Center, Boston, Mass.).
Indirect immunofluorescence.
COS-7 transfectants were plated on coverslips for 3 h before overnight stimulation with EGF (100 ng/ml). In some cases, cells were incubated in the presence of Gö6976 (1 µM), a conventional PKC inhibitor (23). The cells were then fixed using 1% paraformaldehyde in PBS for 20 min, followed by permeabilization for 10 min using 0.5% Triton X-100 in PBS. The cells were rinsed with PBS and blocked and double stained as described previously (34). The preparations were analyzed using standard fluorescence microscopy. A quantitative analysis was performed by counting the number of cells showing hemidesmosomal plaques as a percentage of the total number of cells expressing similar levels of 6ß4.
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RESULTS |
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6 subunit, which is also immunoprecipitated by the ß4 antibody, was not observed (data not shown). The radiolabeled bands corresponding to the ß4 subunit were excised and subjected to phosphoamino acid analysis. As shown in Fig. 1B, the bulk of the phosphorylation induced by EGF was on serine (96%) with significantly less phosphorylation on tyrosine and threonine (3% and 1%, respectively), a pattern similar to that observed for EGF stimulation of A431 cells (35). Subsequently, phosphopeptide mapping was performed by digesting the excised ß4 band with trypsin and resolving the sample by using TLE in one dimension and TLC in the second dimension. This analysis yielded approximately 20 discernible phosphopeptides from both HaCat cells (Fig. 2A [only 10 are labeled for reference]) and A431 cells (data not shown). Most of the radioactivity (
50%), however, was contained in three phosphopeptides (pp0, pp1, and pp2, below the reference line in Fig. 2A). The spatial relationship (i.e., diagonal laddering) of these three peptides suggested that they are isoforms of the same multilabeled phosphopeptide (5).
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-chymotrypsin). As shown in Table 1, pp1 and pp2 were sensitive to V8 protease (cuts the E-X bond, where X is any amino acid) and Asp-N-endoproteinase (cuts the X-D bond) but not chymotrypsin (cuts the F-X, W-X, or Y-X bond). In contrast, the ppf peptide, used as a control, was digested by -chymotrypsin but not the other proteases.
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-chymotrypsin (no F, W, or Y in either candidate). To assess the validity of the identified sequences, site-specific mutagenesis and subsequent biochemical analyses were performed. Initially, we mutated (underlined residues below) the first serine (S1356 or S1427 SerAla or SerAsp) of the two candidate phosphopeptides (PP-I and PP-II) within the full-length ß4 cDNA (Fig. 4B). The ß4 wild-type, ß4S1356A (PP-I), and ß4S1427A (PP-II) plasmids were transiently transfected into COS-7 cells, and 2 days later, the cells were metabolically labeled with 32PO4, stimulated with EGF, and processed for peptide mapping. The phosphopeptide map of wild-type ß4 was very similar to that observed for HaCat and A431 cells (shown for HaCat in Fig. 2A). Analysis of SerAla mutants at the first position of PP-I and PP-II revealed that pp0 disappeared only for the PP-I (ß4S1356A in Fig. 2B and 4B) and not for PP-II (Fig. 4B). Moreover, an alteration in the mobility of pp1 and pp2 from ß4S1356A (PP-I) towards more hydrophobic regions was observed, consistent with the gain of hydrophobicity predicted for a multiphosphorylated peptide that has one of its serines mutated to alanine. These results suggested that PP-I is the candidate phosphopeptide and rule out PP-II.
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Asp mutations. Potential phosphorylation of S1358 or S1366 was excluded because triple-mutant constructs containing these mutations, either as S1356 S1358 S1360 or S1356 S1360 S1366, failed to eliminate all three peptides of interest (Fig. 4B). In contrast, the triple mutation of S1356 S1360 S1364 eliminated all three phosphopeptides (Fig. 2D, ß4S1356D S1360D S1364D; and Fig. 4B). Taken together, these results strongly indicate that S1356, S1360, and S1364, which are located in the connecting segment (Fig. 4C), are the major residues phosphorylated on ß4 after EGF stimulation.
PKC- phosphorylates pp1 and pp2 in vitro.
We had shown previously that PKC- is involved in EGF-induced serine phosphorylation of the ß4 integrin subunit, although we did not assess whether it phosphorylated ß4 directly (35). Two of the identified phosphorylation sites, S1360 and S1364, are located within a consensus sequence for PKC (Prosite) (32). Therefore, we examined the possibility that these serines are directly phosphorylated by PKC using immune complexes of ß4 obtained from 6ß4-transfected COS-7 cells as substrates for purified PKC-. As shown in Fig. 5A, no significant phosphorylation of ß4 was seen in the absence of enzyme but the addition of PKC- resulted in significant phosphorylation. Other PKC isoforms were able to phosphorylate ß4, albeit to a lesser extent (0.15x, 0.13x, and 0.40x for PKC-ßI, -ßII, and -, respectively; Fig. 5A), suggesting isoform preference.
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, we used a ß4 construct (ß41392D1356 D1360 D1364) that carries a triple SerAsp mutation on a truncated ß4 (residues 1 to 1392) to perform immune-complex kinase assays with PKC- and subsequent phosphopeptide analysis. The truncation was used to reduce the number of unrelated peptides. As a control, we used the truncated cDNA lacking the serine mutations (ß41392). The peptide-mapping analysis revealed that pp1 and pp2 are present in the control construct but eliminated by the triple mutation (Fig. 5B). These results indicate that at least two of the three major serines are phosphorylated by PKC-, most likely S1360 and S1364, because they are located within a consensus PKC site (Prosite) (32). Conventional PKC members prefer substrates with basic residues at positions –6, –4, –2, +2, or + 3 and a hydrophobic residue at + 1 (32).
Serine phosphorylation on S1356, S1360, and S1364 mediates the effects of EGF on hemidesmosomal plaques.
COS-7 cells have been used as a model to study the formation of type II hemidesmosomes because they express plectin and intermediate filaments but lack 6ß4 (27). Expression of this integrin results in the recruitment of plectin into "plaque-patterned" hemidesmosomes (27) which can be detected by colocalization of 6ß4 and HD1/plectin using indirect immunofluorescence (Fig. 6A to C). We have previously shown that EGF disrupts hemidesmosomes and induces serine phosphorylation of the ß4 integrin (35). To test whether phosphorylation of serines S1356, S1360, and S1364 mediates the disruptive action of EGF on hemidesmosomes, we expressed single, double, or triple SerAla mutations (ß4S1356A, ß4S1360A, ß4S1356A S1360A, and ß4S1356A S1360A S1364A) in COS-7 cells, predicting that these mutations would prevent hemidesmosome disruption induced by EGF. In the absence of EGF, the mutation of these serines did not significantly alter the ability of exogenous ß4 to form hemidesmosomal plaques (Fig. 7A). In the presence of EGF, the frequency of hemidesmosomal plaque formation by wild-type ß4 was reduced significantly (Fig. 7A), confirming previous results (21, 35). Mutations in S1356, S1360, and S1364, in contrast, impeded the ability of EGF to disrupt hemidesmosomal plaques. The double mutant ß4S1356A S1360A was more effective than the single mutants in this regard. These results suggest that cooperative phosphorylation on S1356, S1360, and S1364 mediates the disruption of type II hemidesmosomes in response to EGF. The fact that the SerAla mutation of these serines did not affect hemidesmosome formation suggests that they are not essential for the formation of the hemidesmosome itself.
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Asp mutants that mimic constitutive phosphorylation in the absence of exogenous stimuli (ß4S1356D, ß4S1360D, ß4S1356D S1360D, and ß4S1356D S1360D S1364D). The ability to form hemidesmosomal plaques differed among these mutants (Fig. 7B). The double and triple phosphorylation-mimicking mutants ß4S1356D S1360D and ß4S1356D S1360D S1364D exhibited a substantial reduction in the plaque pattern, although some recruitment of plectin was still observed in threadlike structures (Fig. 6D to F and Fig. 7B). Expression of the single mutations did not significantly differ from that in the wild type. These results indicate that cooperative phosphorylation on S1356, S1360, and S1364 inhibits hemidesmosome formation.
The major ß4 serine phosphorylation sites (S1356, S1360, and S1364) mediate the effects of PKC- on hemidesmosomal plaques.
We evaluated the hypothesis that the inhibition of conventional PKCs either modifies the formation of hemidesmosomal plaques or prevents the disruption of these structures by EGF stimulation. COS-7 cells cotransfected with 6 and ß4 were treated with the conventional PKC inhibitor Gö6976 before stimulation with EGF. As shown in Fig. 8A, this inhibition significantly increased the formation of hemidesmosomal plaques. Moreover, the effects of EGF were substantially, although not completely, reduced by the inhibitor, indicating that conventional PKCs play an important role in the overall stability of the hemidesmosome even in nonstimulated cells. Subsequently, we tested the possibility that PKC- is sufficient to induce the disruption of hemidesmosomal plaques by cotransfecting 6ß4 with a myristylated form of PKC- (myrPKC-) that acts as a constitutively active variant of this isoform. As shown in Fig. 8B, expression of myrPKC- reduced the number of hemidesmosomal plaques substantially. Moreover, mutation of S1356, S1360, and S1364 in ß4 impeded the effects of myrPKC- on hemidesmosomal plaques, indicating that phosphorylation of these serines is important in the overall stability of the plaque.
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DISCUSSION |
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6ß4 integrin, a component of the hemidesmosome, plays a pivotal role in the formation and stability of this multiprotein structure and, as a consequence, the ability of epithelial cells to migrate (4, 24). Previously we and others had shown that EGF, acting as a chemotactic factor, induces mobilization of the 6ß4 integrin and promotes hemidesmosome disassembly (21, 35). EGF stimulates a PKC--dependent pathway that results in the phosphorylation of the ß4 integrin subunit on serine residues and its redistribution to actin-rich structures (35). Subsequent to our work, other groups have supported the notion that PKC mediates the serine phosphorylation of ß4 (1, 37). To understand how EGF induces disassembly of the hemidesmosome, it was essential to identify the main serine phosphorylation sites, assess whether these sites are critical for disassembly, and determine the kinase(s) responsible for their phosphorylation. We now have resolved all of these key issues by showing that EGF induces the phosphorylation of the ß4 subunit on a cluster of serines (S1356, S1360, and S1364) located within the connecting segment of the cytoplasmic tail. The combined phosphorylation of S1356, S1360, and S1364 is significant quantitatively because it accounts for 50% of the total phosphorylation of ß4 after EGF stimulation. Moreover, the fact that these serines are evolutionarily conserved in the sequences of human, rat, and mouse ß4 (10, 17, 42) suggests they are critical for the function of the 6ß4 integrin.
The cytoplasmic tail of ß4 is essential for both hemidesmosome formation and disassembly (8, 31, 38, 40). Our results substantiate the importance of serine phosphorylation of the ß4 cytoplasmic tail in hemidesmosome disassembly. Specifically, we demonstrate that the ability of EGF to disrupt hemidesmosomes is reduced significantly by mutations that prevent the phosphorylation of S1356, S1360, and S1364. Double or triple mutations of these serines were more efficient than were single mutations in preventing EGF action on hemidesmosomes, suggesting that there is cooperation among these phosphorylation sites to regulate hemidesmosome disassembly. S1356, S1360, and S1364 are located immediately after the minimal region considered necessary for the recruitment of HD1/plectin to the hemidesmosome, and preventing the phosphorylation of S1356, S1360 and S1364 by mutating these residues to alanine had no significant effect on the ability of 6ß4 to recruit plectin and form hemidesmosomal plaques. This result is consistent with a previous study demonstrating that a truncated form of ß4 (nucleotides 1 to 1355) that excludes the serine cluster is still capable of recruiting HD1/plectin (27). The sustained phosphorylation of S1356, S1360, and S1364, however, probably affects the formation or the stability of the hemidesmosome, as evidenced by the reduced number of hemidesmosomal plaques observed for the phosphorylation-mimicking mutants. This effect also appears to involve cooperation of phosphorylation of the three serine residues because the double and triple SerAsp mutations were more effective than the single mutations at reducing hemidesmosomal plaques.
Although mutations in the major serine phosphorylation sites that we identified had a significant effect on hemidesmosome dynamics, none of these mutants resulted in complete elimination of hemidesmosomes and recruitment of plectin. This observation suggests that additional mechanisms of disruption exist, such as other phosphorylation sites. Previous studies had shown that preventing the phosphorylation of two tyrosines in the connecting segment (Y1422 and Y1440) caused a partial inhibition of hemidesmosome disassembly induced by EGF (7). Taken together, these results suggest that the phosphorylation of multiple residues is needed for complete abrogation of the disruptive effects of EGF on the hemidesmosome. Given that our peptide mapping analyses identified approximately 20 potential phosphorylation sites in the ß4 subunit, this assumption seems valid. Moreover, a complex regulation is also indicated by the multiplicity of interaction sites between ß4 and HD1/plectin (27, 28, 30, 36). The ß4 integrin has at least two different binding sites for HD1/plectin (36), and it is reasonable to expect that additional phosphorylation sites might be located throughout the molecule, probably at the carboxy terminus where the second HD1/plectin site is located (36).
The biochemical mechanisms by which phosphorylation of S1356, S1360, and S1364 induce hemidesmosome disruption merit discussion. In addition to a potential inhibition of plectin/ß4 association, phosphorylation of S1356, S1360, and S1364 could also destabilize the hemidesmosome by interfering with ß4 self-association. Both intermolecular and intramolecular interactions of ß4 involving the connecting segment have been reported (36, 38) and could be influenced by phosphorylation of the serine cluster. Disruption of intermolecular interactions by serine phosphorylation may reduce plaque formation. On the other hand, intramolecular interactions may affect the recruitment of other hemidesmosomal components (18, 38). The folding of the carboxy terminus of ß4 onto the connecting segment has been hypothesized to regulate the recruitment of BPAG1 and BPAG2 into type I hemidesmosomes, a process that is facilitated by the binding of HD1/plectin (18). It will be interesting to address whether S1356, S1360, and S1364 regulate the recruitment of BPAG1 and BPAG2 directly or indirectly by regulating ß4-plectin interactions.
The destabilization of the hemidesmosome by the phosphorylation of S1356, S1360, and S1364 could result from conformational changes caused by the electrostatic charges of the added phosphate groups. This possibility is evidenced by the SerAsp mutations that increased the number of charged residues and resulted in hemidesmosome instability. Alternatively, serine phosphorylation may provide the binding site for other destabilizing molecules. It is interesting to note in this regard that other authors have observed PKC--mediated serine phosphorylation on unknown residues in the connecting segment that induces the association of 14-3-3 proteins and, in concert with signals from the Ron receptor, may contribute to the destabilization of the hemidesmosome (37). Ron may also induce tyrosine phosphorylation of ß4 through src-fyn, a pathway also implicated in EGF-mediated disassembly of hemidesmosomes (22).
A key finding in this study is that PKC- phosphorylates the ß4 subunit directly and that at least two of the three serines we identified are substrates for this kinase. These findings are consistent with several observations including our previous study implicating PKC- in the EGF-induced phosphorylation of ß4 (35), the fact that EGF can activate PKC through the phospholipase C--mediated formation of diacylglycerol and inositol triphosphate (15, 33), and a recent study demonstrating that PKC- phosphorylates the connecting segment of ß4 (37). Our study does not rule out the possibility, however, that other kinases might be involved in the phosphorylation of the identified serines as well. For example, PKC- has been shown to phosphorylate ß4 (1). Consistent with this study, we observed that PKC- phosphorylates ß4 in vitro, although to a lesser extent than PKC-. In contrast to our study and that by Santoro et al. (37), a study by Alt et al. (1) was unable to detect ß4 phosphorylation with PKC- or show its disruptive effects on hemidesmosomes. One explanation for the latter finding is that these authors overexpressed wild-type PKC- instead of a constitutively active form. Some studies have shown that the cellular effects produced by overexpression of PKC- may depend on the presence of modulators, such as phorbol myristate acetate (26).
Previous studies have shown that hemidesmosomes type I (6ß4, plectin, and BPAG1 and -2) impede the migration of stratified epithelial cells and that the PKC-mediated disruption of these structures facilitates migration (7, 22, 35). A431 cells, for example, assemble type I hemidesmosomes, and their migration closely correlates with hemidesmosome disassembly induced by EGF (35). Hemidesmosome disassembly and migration of these cells require PKC- activation and are associated with serine phosphorylation of the ß4 subunit (35). Interestingly, we demonstrate in the present study that S1356, S1360, and S1364 are the major sites of EGF-stimulated ß4 serine phosphorylation in A431 cells. This result links phosphorylation of these key serine residues with migration. In contrast, however, we also observed that COS-7 cell expression of the triple-serine mutation of ß4, which resists the disruption of the hemidesmosomal plaque (type II 6ß4 and plectin) after EGF stimulation, did not affect migration (data not shown). This result raises the possibility that the anchor provided by type II hemidesmosomes is different from that of type I hemidesmosomes in terms of resisting migration, the latter being more efficient because it contains at least two more proteins. This assessment is consistent with the fact that cells that normally express type II hemidesmosomes, such as intestinal epithelial cells, are constantly migrating (2).
In summary, we have identified a cluster of serines in the ß4 integrin subunit that are targeted directly by PKC- in response to growth factor stimulation and whose phosphorylation status regulates the dynamics of hemidesmosomes. This mechanism has important implications for the function of the 6ß4 integrin in migration and carcinoma invasion.
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ACKNOWLEDGMENTS |
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We thank Kaylene Simpson, Elizabeth Lipscomb, and Leslie Shaw for valuable discussions.
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FOOTNOTES |
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REFERENCES |
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