GROWTH-HORMONE AND PHORBOL ESTERS REQUIRE SPECIFIC PROTEIN-KINASE-C ISOFORMS TO ACTIVATE MITOGEN-ACTIVATED PROTEIN-KINASES IN 3T3-F442A CELLS

Previous studies have shown that the activation of p44 and p42 mitogen -activated protein (MAP) kinases (ERK1 and ERK2) by growth hormone (GH ) and phorbol esters, but not by epidermal growth factor, in 3T3-F442A preadipocytes is dependent on protein kinase C (PKC). In the present study two approaches have been taken to determine the PKC isoform depe ndence of MAP kinase activation in these cells, By immunoblotting with specific antibodies, the cells were found to express PKC-alpha, -gamm a, -delta, -epsilon and -zeta. Treatment of cells with 500 nM PMA for 3 h led to the complete depletion of PKC-delta and the partial depleti on of PKC-alpha but did not significantly affect the expression of the other PKC isoforms, In parallel, such treatment severely attenuated t he ability of GH to activate MAP kinase, The degree of this attenuatio n was not increased by more prolonged PMA pretreatment, indicating tha t PKC-delta and perhaps PKC-alpha are important for MAP kinase activat ion by GH, These experiments further revealed that additional PI(C iso forms were required for the full activation of MAP kinases by acute tr eatment with PMA. A second approach involved the use of anti-sense oli godeoxynucleotides (ODNs) to deplete the individual PKC isoforms selec tively. Each of the ODNs used effectively depleted the relevant isofor m to undetectable levels and did not affect the expression of the othe r PKC isoforms. Pretreatment of cells with PKC-delta anti-sense ODN, b ut not with anti-sense ODN to the other phorbol ester-sensitive isofor ms, severely attenuated the activation of MAP kinases by GH. PKC-delta anti-sense ODN also blocked (by approx. 50%) the activation of MAP ki nases by PMA. Furthermore a combination of PKC-delta and -epsilon anti -sense ODNs completely blocked the effect of PMA on MAP kinases. Colle ctively, these results indicate that the novel PKC-delta and -epsilon isoforms can couple to the MAP kinase pathway in 3T3-F442A cells but t hat the activation of MAP kinases by GH specifically involves PKC-delt a.