Abstract
The overexpression of protein kinase C-delta (PKC-delta), but not PKC-epsilon, 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-delta that is responsible for this isotype-specific function, cDNAs that encode reciprocal chimeras of PKC-delta and -epsilon (PKC-delta epsilon and PKC-epsilon delta) were constructed by exchanging regulatory and kinase domains using polymerase chain reaction technology. Both chimeras were stably expressed in 32D cells using the pLTR expression vector and displayed protein kinase activity upon TPA treatment. TPA treatment of L epsilon delta, cells that overexpressed the PKC-epsilon delta 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-delta. In contrast, little effect was seen in L delta epsilon, 32D cells that overexpressed PKC-delta epsilon, with or without TPA treatment. A PKC inhibitor directed toward the catalytic domain of PKC, GF109203X, and a selective inhibitor of PKC-delta, Rottlerin, blocked the TPA-induced differentiation of PKC-epsilon delta-overexpressing 32D cells. These results demonstrate that the catalytic domain of PKC-delta contains the primary determinants for its activity in phorbol ester-induced macrophage differentiation.
Highlights
The overexpression of protein kinase C-␦ (PKC-␦), but not Protein kinase C (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)
Construction of PKC-␦ and -⑀ Chimeras—Before generating the PKC chimeras, we separately amplified the regulatory and catalytic domains of PKC-␦ and -⑀ from their cDNAs using polymerase chain reaction (PCR) and a series of oligonucleotide primer pairs each of which includes a 21-mer that is present in the C3 region of both PKC-␦ and -⑀
The chimeric PKC-␦⑀ was identified as a protein with the size of PKC-␦ that reacted with an antibody against the C terminus of PKC-⑀ but not with one against the C terminus of PKC-␦
Summary
76 –82, 1997 Printed in U.S.A. The Catalytic Domain of Protein Kinase C-␦ in Reciprocal ␦ and ⑀ Chimeras Mediates Phorbol Ester-induced Macrophage Differentiation of Mouse Promyelocytes*. 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 chimeras were stably expressed in 32D cells using the pLTR expression vector and displayed protein kinase activity upon TPA treatment. PKC-␦ Catalytic Domain Mediates Macrophage Differentiation domain of PKC-␦ was responsible for its ability to confer TPAinduced differentiation on this myeloid cell line
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