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

Abstract

To study enzymatic activity and activation conditions of the recently identified novel protein kinase C ~t (PKC~t) subtype, epitope tagged PKC~t was propagated in the baculovirus expression system and was purified to homogeneity. PKC~t displays high affinity phorbol ester binding (Kd = 7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and Ptdlns-4,5-P2, were found to promote PKCp~ 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 PKC~t 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 PKC~t.

Inhibition of protein kinase C μ by various inhibitors. Inhibition from protein kinase c isoenzymes

“…Sf158 cells were infected with a recombinant PKCIa baculovirus as described [11]. Cells were lysed with buffer A (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 mM ~-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 3 mg/ml leupeptin).…”

“…is Ca2+-independent and activated by phorbol ester [6,11], one would tend to associate it with the nPKC group of PKC isoenzymes. However, the excellent inhibition by G6 6976, which is atypical for an nPKC, as well as various structural and functional properties (see [5,6,10,11]) suggest that PKCkt might represent a novel type that does not fit into any of the known PKC subgroups. Other staurosporine-derived compounds, the bisindolylmaleimides G6 6983, bisindolylmaleimide I (GF-109203X) and bisindolylmaleimide III (for chemical structures see Fig.…”

“…This prompted us to look for a putative pseudosubstrate motif of PKCB. In this context, we tested three synthetic PKC B peptides as potential inhibitors of PKC B. Peptide 1 (4°'SPLMRVVQSVKHTKRK 419) was chosen, since it displays some homology to syntide 2, a known in vitro substrate of PKCIa [11]. The peptides 2 (75AHVREMACSIVDQK88) and 3 (>~2FQIRPHALFVHSY-RAPAF159), like the known PKC pseudosubstrate sequences, are located N-terminal of the two cysteine-rich regions and contain the consensus sequence RXXA that appears to be an essential part of the pseudosubstrate motifs of the other PKC isoenzymes.…”

“…It was reported that PKD failed to phosphorylate several substrates which are actively phosphorylated by other members of the PKC family. The only substrate for PKCp./PKD known so far are the synthetic peptides syntide 2 and GS [10,11] According to its structural and functional properties, PKCpJPKD appears to be a member of the PKC family but cannot be included in any of the three subgroups. Several reports have indeed indicated the involvement of PKC isoenzymes in specific cellular processes, like differentiation (PKC~i) and proliferation (PKCe) of specific cells (for a review see [3]).…”

Basic Calcium Phosphate Crystals Activate p44/42 MAPK Signal Transduction Pathway via Protein Kinase Cμ in Human Fibroblasts

“…Autophosphorylation was started by adding 30 l of the phosphorylation buffer containing 0.3 l of [␥-32 P]ATP (5000 Ci/mmol and 10 mCi/ ml) and L-␣-phosphatidyl-L-serine (100 g/ml) to the immunocomplexes and incubating at 37°C for 20 min (23,25,32). The reactions were terminated by adding an equal volume of 2ϫ SDS sample buffer to each one.…”