PKCE-Related Kinase Associates with and Phosphorylates Cytokeratin 8 and 18

A 40-kD protein kinase C (PKC)E related activity was found to associate with human epithelial specific cytokeratin (CK) polypeptides 8 and 18 . The kinase activity coimmunoprecipitated with CK8 and 18 and phosphorylated immunoprecipitates of the CK. Immunoblot analysis of CK8/18 immunoprecipitates using an anti-PKCE specific antibody showed that the 40-kD species, and not native PKCE (90 kD) associated with the cytokeratins . Reconstitution experiments demonstrated that purified CK8 or CK18 associated with a 40-kD tryptic fragment of purified PKCE, or with a similar species obtained from cells that express the fragment constitutively but do not express CK8/18. CYTOKERATINS (CK)' are a group of intermediate filament (IF) proteins which are expressed primarily in epithelial tissues (Lazarides, 1982 ; Steinert and Roop, 1988 ; Franke et al ., 1981 ; Osborn and Weber, 1986) . The 30 or so polypeptides which make up the family ofcytokeratin proteins are divided into acidic (type I) and basic/ neutral (type II) keratins . In epithelial cells, CK are found as mosaic noncovalent polymers with an assembly consisting of at least one type I and one type II CK (Steinert and Roop, 1988) . For example, "simple" single layer epithelial cells such as intestinal epithelia express CK8 (type II) and CK18 (type I), whereas esophageal epithelial cells express CK4 (type II) and CK13 (type I) predominantly. Cytokeratins are not only important as tissue-specific markers, they also form important markers of cell differentiation . IF proteins, including cytokeratins, undergo several posttranslational modifications such as N112-terminal acetylation (Steinert and Idler, 1975), glycosylation (King and Hounsell, 1989 ; Roberts and Brunt, 1986), and serine/threonine phosphorylation (Steinert, 1988 ; Gilmartin et al ., 1984 ; Yeagle et al ., 1990 ; Baribault et al ., 1989) . The functional role of IF protein phosphorylation is not well under1 . Abbreviations used in thispaper: CK, cytokeratins; IEF, isoelectric focusing ; IF, intermediate filament; PKC, protein kinase C ; SCLC, small cell lung carcinoma . © The Rockefeller University Press, 0021-9525/92/05/583/11 $2.00 The Journal of Cell Biology, Volume 117, Number 3, May 1992 583-593 A peptide pseudosubstrate specific for PKCE inhibited phosphorylation of CK8/18 in intact cells or in a kinase assay with CK8/18 immunoprecipitates . Tryptic peptide map analysis -of the cytokeratins that were phosphorylated by purified rat brain PKCE or as immunoprecipitates by the associated kinase showed similar phosphopeptides . Furthermore, PKCE immunoreactive species and CK8/18 colocalized using immunofluorescent double staining . We propose that a kinase related to the catalytic fragment of PKCE physically associates with and phosphorylates cytokeratins 8 and 18. stood but phosphorylation of vimentin, desmin, neurofilaments, and nuclear lamins appears to be important in regulating the assembly of these filaments during mitosis (Inagaki et al ., 1987, 1988 ; Gonda et al ., 1990) . The kinases that are involved in CK phosphorylation have not been identified although evidence exists that CAMP-dependent protein kinase can phosphorylate several cytokeratins (Steinert, 1988 ; Gilmartin et al ., 1984) . In addition, protein kinase C (PKC) appears to play a role in CK8/18 phosphorylation (Chou and Omary, 1991) . PKC is a serine/threonine protein kinase with important functional roles in the cellular responses ofmany tissues (for reviews see Nishizuka 1988, 1989 ; Parker et al ., 1989 ; Bell and Burns, 1991) . At least eight subspecies ofPKC have been identified (tx, ßI, 011, y, S, E, r, and rl) with differences in structure, substrate, and calcium dependence of the PKC subspecies described (Nishizuka, 1988 ; Osada et al ., 1990 ; Bacher et al ., 1991) . PKCE (90 kD), is a calcium-independent member ofthe PKC family with a broad tissue distribution as determined by Northern blot analysis of rat tissue mRNA (Ono et al ., 1988 ; Schaap et al ., 1989), or by Western blot analysis using specific PKCE antisera (Strulovici et al ., 1991) . Limited in vitro proteolytic digestion of PKC with trypsin or calpain results in the generation of a stable fragment of 40-50 kD, (Kishimoto et al . , 1989 ; Schaap et al . , 1990 ; 583 on M ay 2, 2017 D ow nladed fom Published May 1, 1992