[PMC free article] [PubMed] [Google Scholar]Boyle W. They guarantee stable adhesion of these cells to the underlying basement membrane. HDs are present in complex and (pseudo) stratified epithelia and are composed of at least six proteins: the two subunits of the integrin 64 (Stepp (2004) showed that this serine residues S1356, S1360, and S1364 in the 4 CS are phosphorylated downstream of the EGF receptor, and they suggest that this is mediated by PKC. Moreover, substitution of these three residues by aspartic acid results in the partial loss of the colocalization of plectin with 64 in transfected COS-7 cells. Therefore, we decided to investigate the role of these serines in the disassembly of HDs under more physiological conditions in keratinocytes. By phosphatase inhibition assays three regions of 4 were recognized that were greatly phosphorylated on serine and cIAP1 ligand 1 threonine residues in cells, including the region of the CS made up of S1356, S1360, and S1364. We further show that activation of endogenous PKC results in the phosphorylation of only S1360. We also show through phosphomimicry studies that phosphorylation of two or more of these serines prevents the primary interaction of the plectin ABD with 4. Furthermore, we recognized S1364 as an endogenous PKA phosphorylation site in keratinocytes and showed that its activation in conjunction with PKC reduces the ability of the plectin ABD to associate with 4. And finally, we established that activation of the EGF receptor results in the phosphorylation of only these three residues in keratinocytes, which leads to a partial disassembly cIAP1 ligand 1 of HDs. Taken together, our results suggest that in keratinocytes, multiple kinases take action in concert to induce the disassembly of HDs through the phosphorylation of S1356, S1360, and S1364. MATERIALS AND METHODS Cell Lines PA-JEB (4-null) keratinocytes have been explained previously (Schaapveld cIAP1 ligand 1 DNA polymerase (Roche Molecular Biochemicals, Indianapolis, IN) was used. All plasmids were verified by sequencing, and protein expression and sizes were confirmed by Western blotting. The full-length 4 cDNA in pUC18 has been explained previously (Niessen and show the amount of colocalization between 6 and plectin. In the right panels, high intensity colocalizing pixels are shown in blue, noncolocalized high intensity 6 pixels are shown in reddish, whereas noncolocalized high-intensity plectin pixels are shown in yellow. Pulldown and Phospho-specific Antibody Assays COS-7 cells were transfected with indicated cDNA constructs. When indicated, COS-7 or PA-JEB/4 cells were stimulated with either 1 M PMA (Sigma-Aldrich), 25 M FSK (Calbiochem), and 0.1 mM IBMX (Calbiochem) or 50 ng/ml EGF (Sigma-Aldrich) for 30 min (or for the indicated occasions in cIAP1 ligand 1 the time-course stimulation assays) at 37C. Cells were lysed in M-PER buffer (Mammalian Protein Extraction Reagent; Pierce), made up of 1 mM sodium vanadate and a cocktail of protease inhibitors (Sigma-Aldrich). Immunoprecipitations were performed with mAb HA 12CA5 (Santa Cruz Biotechnology, Santa Cruz, CA) and immunoblottings were done with either pAb anti-IL2R (sc-665; Santa Cruz Biotechnology), pAb anti-HA (sc-805; Santa Rabbit polyclonal to LPA receptor 1 Cruz Biotechnology), mAb anti-pTyr (4G10), pAb anti-EGF receptor (Ab-17; Neomarkers or 1005; Santa Cruz Biotechnology), mAb anti-EGFR pY845 (Cell Signaling, Beverly, MA), a mixture of mAbs antibodies against PKC, cIAP1 ligand 1 , and (Transduction Laboratories, Lexington, KY) or pAb anti-phospho-PKC (pan; betaII Ser 660; 9371; Cell Signaling) as explained previously (Litjens (2004) have shown that substitution of three serine residues at positions 1356, 1360, and 1364 in 4 by phosphomimic aspartic acid residues impairs formation of HDs in transfected COS-7 cells..