In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase Cμ

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Abstract
To study enzymatic activity and activation conditions of the recently identified novel protein kinase C μ (PKCμ) subtype, epitope tagged PKCμ was propagated in the baculovirus expression system and was purified to homogeneity. PKCμ displays high affinity phorbol ester binding (K d = 7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns-4,5-P2, were found to promote PKCμ kinase activity. Two peptides derived from the glycogen synthase, GS-peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine-rich C-kinase substrate) served as an in vitro substrate for PKCμ too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKCμ.