The overexpression of protein kinase C-␦ (PKC-␦), but not PKC-⑀, enables the mouse myeloid cell line 32D to differentiate into macrophages when treated with phorbol esters such as 12-O-tetradecanoylphorbol-13-acetate (TPA). To determine the domain of PKC-␦ that is responsible for this isotype-specific function, cDNAs that encode reciprocal chimeras of PKC-␦ and -⑀ (PKC-␦⑀ and PKC-⑀␦) were constructed by exchanging regulatory and kinase domains using polymerase chain reaction technology. Both

more »

... meras were stably expressed in 32D cells using the pLTR expression vector and displayed protein kinase activity upon TPA treatment. TPA treatment of L⑀␦, cells that overexpressed the PKC-⑀␦ chimera, induced a dramatically increased cell volume, surface adherence, surface expression of Mac-1 and Mac-3, lysozyme production, and phagocytosis. These are the characteristics of the macrophage phenotype found in TPA-treated 32D cells that overexpressed PKC-␦. In contrast, little effect was seen in L␦⑀, 32D cells that overexpressed PKC-␦⑀, with or without TPA treatment. A PKC inhibitor directed toward the catalytic domain of PKC, GF109203X, and a selective inhibitor of PKC-␦, Rottlerin, blocked the TPA-induced differentiation of PKC-⑀␦overexpressing 32D cells. These results demonstrate that the catalytic domain of PKC-␦ contains the primary determinants for its activity in phorbol ester-induced macrophage differentiation.