PROTEIN-KINASE C-BETA-1 AND PROTEIN-KINASE C-BETA-2 ACTIVATE P57 MITOGEN-ACTIVATED PROTEIN-KINASE AND BLOCK DIFFERENTIATION IN COLON-CARCINOMA CELLS

When HD3 colon carcinoma cells differentiate to fluid-transporting, en terocytic-like cells, they down-regulate their protein kinase C (PKC) beta levels 5-10-fold and lose two responses to basic fibroblast growt h factor (FGF): proliferation and the ability to activate p57 mitogen- activated protein (MAP) kinase, HD3 cells were transfected with expres sion plasmids for the splice variants PKC-beta 1 and PKC-beta 2 and th e empty vector for a control, Each of two PKC-beta 1 and each of two P KC-beta 2 transfectant clones exhibited elevated levels of Ca2+- and p hosphatidylserine-dependent PKC activity, Both PKC-beta 1 transfectant clones had elevated levels of PKC-beta 1 protein compared with the PK C-beta 2 transfectants or controls, whereas both PKC-beta 2 transfecta nt clones had elevated levels of PKC-beta 2 protein compared with PKC- beta 1 transfectants, Control transfectants had no detectable PKC-beta 2 protein, Similar levels of PKC-alpha were found in all lines. Each PKC-beta transfectant was less differentiated than the parental line a nd had regained proliferative response to basic FGF, Increased growth rates in athymic mice were seen for PKC-beta 2 and PKC-beta 1 transfec tant cells, Immunocytochemistry of the sectioned tumors showed enhance d protein levels of PKC-beta 2 and PKC-beta 1, correlating increased l evels of these isozymes with increased growth, Increased myelin-basic protein (MBP) kinase activities of M(r) 44,000, 57,000, 63,000, 110,00 0, and 130,000 by in-gel kinase assay characterized each PKC-beta tran sfectant. Both Western blotting and immunoprecipitation studies from S -35- prelabeled cells with a pan-erk antibody showed no increase in pr otein abundance of MAP kinases of M(r) 44,000, 57,000, and 63,000, sug gesting that elevated PKC-beta levels led to activation of the smaller three MAP and MBP kinases. Activation of p57 MAP kinase in each PKC-b eta transfectant was demonstrated by immunoprecipitation with an antip hosphotyrosine monoclonal antibody and then by assay of the immunoprec ipitates by in-gel kinase assay on MBP, p57 MAP kinase was distinguish ed from the M(r) 54,000 stress-activated protein kinases, which migrat ed more rapidly on SDS gels and could be detected by in-gel kinase ass ay on MBP only after cellular stress. Thus, expression of elevated lev els of PKC-beta 1 and PKC-beta 2 in differentiated HD3 colon carcinoma cells blocked their differentiation, enabled them to proliferate in r esponse to basic FGF like undifferentiated cells, increased their grow th rate in athymic mice, and activated several MBP kinases, among them , p57 MAP kinase.