Vol. 7, 587-594, May 1996 Cell Growth & Differentiation 587 Protein Kinase Cr31 and Protein Kinase C2 Activate p57 Mitogen-activated Protein Kinase and Block Differentiation in Colon Carcinoma Cells1 Samir Sauma, Zhongfa Van, Shigeo Ohno, and Eileen Friedman2 Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [S. S., Z. Y., E. F.), and Yokahama City University School of Medicine, Fuku-ura, Kanazawa-ku, Yokohama 236, Japan [S. 0.] Abstract When HD3 colon carcinoma cells differentiate to fluid- transporting, enterocytic-like cells, they down-regulate their protein kinase C (PKC) 1 levels 5-10-fold and lose two responses to basic fibroblast growth factor (FGF): proliferation and the ability to activate p57 mitogen- activated protein (MAP) kinase. HD3 cells were transfected with expression plasmids for the splice variants PKC- 31 and PKC-132 and the empty vector for a control. Each of two PKC-1J1 and each of two PKC- fi2 transfectant clones exhibited elevated levels of Ca2 - and phosphatidylserine-dependent PKC activity. Both PKC-fJl transfectant clones had elevated levels of PKC-fil protein compared with the PKC-fi2 transfectants or controls, whereas both PKC- 32 transfectant clones had elevated levels of PKC-fi2 protein compared with PKC- 1 transfectants. Control transfectants had no detectable PKC-fi2 protein. Similar levels of PKC-a were found in all lines. Each PKC-fi transfectant was less differentiated than the parental line and had regained proliferative response to basic FGF. Increased growth rates in athymic mice were seen for PKC-fi2 and PKC-131 transfectant cells. Immunocytochemistry of the sectioned tumors showed enhanced protein levels of PKC- 2 and PKC-fil, correlating increased levels of these isozymes with increased growth. Increased myelin-basic protein (MBP) kinase activities of Mr 44’000’ 57,000, 63’000’ 1 10,000, and 130,000 by in-gel kinase assay characterized each PKC-fi transfectant. Both Western blotting and immunoprecipitation studies from as5 prelabeled cells with a pan-cit antibody showed no increase in protein abundance of MAP kinases of M 44,000, 57,000, and 63,000, suggesting that elevated PKC-fi levels led to activation of the smaller three MAP Received 10/6/95; revised 2/8/96; accepted 2/22/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1 734 solely to mdi- cate this fact. 1 This work was supported by National Cancer Institute Grants AOl CA45783 and AOl CA67405 (E. F.). 2 To whom requests for reprints should be addressed, at Department of Pathology, State University of New York Syracuse Health Science Center, 750 E. Adams Street, Syracuse, NY 13210. and MBP kinases. Activation of p57 MAP kinase in each PKC- 3 transfectant was demonstrated by immunoprecipitation with an antiphosphotyrosine monoclonal antibody and then by assay of the immunoprecipitates by in-gel kinase assay on MBP. p57 MAP kinase was distinguished from the M 54’000 stress-activated protein kinases, which migrated more rapidly on SDS gels and could be detected by in-gel kinase assay on MBP only after cellular stress. Thus, expression of elevated levels of PKC-fil and PKC-j32 in differentiated HD3 colon carcinoma cells blocked their differentiation, enabled them to proliferate in response to basic FGF like undifferentiated cells, increased their growth rate in athymic mice, and activated several MBP kinases, among them, p57 MAP kinase. Introduction p57 MAP3 kinase is antigenically related to the most com- monly known MAP kinases, erks i-3 (i , 2). p57 MAP kinase, like erks i and 2 (3-5), is activated severalfold as a kinase by growth factors and PKC activators, which increase its thre- onine and tyrosine phosphorylation (6, 7). p57 MAP kinase is activated in colon carcinoma cells by growth factors that also activate erki , although the basal kinase activity of erki seems much lower (6, 7). Both MAP kinases may play dif- ferent roles in mitogenesis, possibly by having different tar- gets. We had observed earlier that differentiated colon car- cinoma cells that had down-regulated PKC-f3 abundance and activity were incapable of activating p57 MAP kinase (6). Each of two independently cloned colon carcinoma lines that displayed differentiation characteristics of mature fluid- transporting colon enterocytes had down-regulated Ca2 - and phosphatidylserine-dependent PKC activity, specifically PKC-j3 (5-i 0 fold), compared with each of two undifferenti- ated lines capable of transmitting mitogenic signals to p57 MAP kinase. Both differentiated and undifferentiated lines maintained equal levels of the PKC isozymes a, a, and , and none of the four lines exhibited either PKC-’y or PKC-8 (6), limiting at least the major, if not the entire, PKC modulation to the PKC-p isozyme. In the current study, we transfected both splice variants of PKC-p (8), PKC- 3i and PKC-132, into one of these differentiated lines and determined the effect of increased PKC- i and PKC- 2 expression on cell differen- tiation and the capacity to activate p57 MAP kinase. 3 The abbreviations used are: MAP, mitogen-activated protein; PKC, pro- tein kinase C; FGF, fibroblast growth factor; MBP, myelin basic protein; SAPK, stress-activated protein kinase; TPA, i2-0-tetradecanoylphorbol- 13-acetate; PVDF, polyvmnylidene difluoride; PMSF, phenylmethylsulfonyl fluoride.