When
HD3 colon
carcinoma cells differentiate to fluid-transporting, enterocytic-like cells, they down-regulate their
protein kinase C (
PKC) beta 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-beta 1 and
PKC-beta 2 and the empty vector for a control. Each of two
PKC-beta 1 and each of two
PKC-beta 2 transfectant clones exhibited elevated levels of Ca(2+)-and
phosphatidylserine-dependent PKC activity. Both
PKC-beta 1 transfectant clones had elevated levels of
PKC-beta 1 protein compared with the
PKC-beta 2 transfectants or controls, whereas both
PKC-beta 2 transfectant 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 and had regained proliferative response to basic FGF. Increased growth rates in athymic mice were seen for
PKC-beta 2 and
PKC-beta 1 transfectant cells. Immunocytochemistry of the sectioned
tumors showed enhanced
protein levels of
PKC-beta 2 and
PKC-beta 1, correlating increased levels of these isonzymes 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-beta 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 M(r) 44,000, 57,000, and 63,000, suggesting that elevated
PKC-beta levels led to activation of the smaller three MAP and MBP
kinases. Activation of p57 MAP
kinase in each
PKC-beta 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-beta 1 and
PKC-beta 2 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.