| Abstract: |
When HD3 colon carcinoma cells differentiate to fluid-transporting, enterocytic-like cells, they down-regulate their protein kinase C (PKC) β 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-β1 and PKC-β2 and the empty vector for a control. Each of two PKC-β1 and each of two PKC-β2 transfectant clones exhibited elevated levels of Ca2+- and phosphatidylserine-dependent PKC activity. Both PKC-β1 transfectant clones had elevated levels of PKC-β1 protein compared with the PKC-β2 transfectants or controls, whereas both PKC-β2 transfectant clones had elevated levels of PKC-β2 protein compared with PKC- β1 transfectants. Control transfectants had no detectable PKC-β2 protein. Similar levels of PKC-α were found in all lines. Each PKC-β 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-β2 and PKC-β1 transfectant cells. Immunocytochemistry of the sectioned tumors showed enhanced protein levels of PKC-β2 and PKC-β1, correlating increased levels of these isozymes with increased growth. Increased myelin- basic protein (MBP) kinase activities of M(r) 44,000, 57,000, 63,000, 110,000, and 130,000 by in-gel kinase assay characterized each PKC-β transfectant. Both Western blotting and immunoprecipitation studies from 35S-prelabeled cells with a pan-erk antibody showed no increase in protein abundance of MAP kinases of Mr 44,000, 57,000, and 63,000, suggesting that elevated PKC-β levels led to activation of the smaller three MAP and MBP kinases. Activation of p57 MAP kinase in each PKC-β 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(r) 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-β1 and PKC-β2 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. |
