Biochemical characterization of the multifunctional Ca super(2+)/calmodulin-dependent protein kinase type IV expressed in insect cells

• Published in: The Journal of biological chemistry 1993-01, Vol.268 (35), p.26171-26178
• Main Authors: Cruzalegui, F H, Means, A R
• Format: Article
• Language: English
• Subjects: Insecta
• ISSN: 0021-9258

We have expressed the rat brain Ca super(2+)/calmodulin (CaM)-dependent protein kinase type IV in insect cells. The recombinant enzyme is produced as a single polypeptide that migrates on SDS-polyacrylamide gel electrophoresis at 61 kDa. Recombinant CaM kinase IV undergoes slow CaM-dependent autophosphorylation. The autophosphorylation of CaM kinase IV occurs on serine residues but is not accompanied by the generation of a CaM-independent activity, as previously reported for the cerebellar enzyme. Comparison of peptide and protein phosphorylation by the recombinant CaM kinase IV and the cerebellar enzyme showed differences in their catalytic activities. The deduced primary sequence of CaM kinase IV contained a domain, super(315)Phe-Asn-Ala-Arg-Arg-Lys-Leu-Lys super(323), also found in the regulatory domain of CaM kinase II alpha (residues 293-300). Truncation of CaM kinase IV at Leu super(313) (at a position analogous to Leu super(290) in CaM kinase II alpha ) generated a fully active, CaM-independent enzyme. This truncated enzyme no longer bound CaM. These data confirm that CaM kinase IV demonstrates intrasteric regulation by an autoinhibitory domain and provides insight into a potentially common mechanism for the regulation of the CaM-dependent multifunctional protein kinases. A number of synthetic peptides were examined for their phosphorylation by both CaM kinase II and IV.