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In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu
S Dieterich1, T Herget, G Link
1Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Abstract:
To study enzymatic activity and activation conditions of the recently identified novel protein kinase C mu (PKC mu) subtype, epitope tagged PKC mu was propagated in the baculovirus expression system and was purified to homogeneity. PKC mu 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 PtdIns-4,5-P2, were found to promote PKC mu 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 mu 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 mu.
Insights
Protein kinase C mu (PKC mu) is a novel enzyme activated by specific lipids and phorbol esters. It phosphorylates substrates like MARCKS differently than other PKC subtypes, suggesting unique regulatory roles.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Protein kinase C mu (PKC mu) is a recently identified member of the PKC family.
- Understanding its enzymatic activity and activation is crucial for elucidating its biological functions.
Purpose of the Study:
- To characterize the enzymatic activity of PKC mu.
- To determine the activation conditions and substrate specificity of PKC mu.
Main Methods:
- PKC mu was expressed using a baculovirus system and purified.
- Enzymatic activity was assessed using various lipid activators and peptide substrates.
- Phorbol ester binding affinity and substrate phosphorylation sites were analyzed.
Main Results:
- Purified PKC mu exhibited high-affinity phorbol ester binding (Kd=7 nM).
- Activation required phosphatidylserine, diacylglycerol, and PtdIns-4,5-P2.
- GS-peptide, syntide 2, and MARCKS were identified as substrates.
- PKC mu phosphorylated MARCKS peptide specifically at serine 156, unlike other PKCs.
Conclusions:
- PKC mu possesses distinct activation requirements and substrate preferences.
- Differential phosphorylation of MARCKS suggests unique regulatory mechanisms mediated by PKC mu.