The Catalytic Domain of Acanthamoeba Myosin I Heavy Chain Kinase

doi:10.1074/jbc.271.43.27056

The Catalytic Domain of Acanthamoeba Myosin I Heavy Chain Kinase

II. EXPRESSION OF ACTIVE CATALYTIC DOMAIN AND SEQUENCE HOMOLOGY TO p21-ACTIVATED KINASE (PAK)*

From the Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892

^‡ To whom correspondence should be addressed:
Bldg. 3, Rm. B1-22, NIH, Bethesda, MD 20892.
Tel.: 301-496-2116 or 1616; Fax: 301-496-1519; E-mail: edk{at}nih.gov

Abstract

Acanthamoeba myosin I heavy chain (MIHC) kinase is a monomeric 97-kDa protein that is activated by binding to acidic phospholipids or by autophosphorylation. Activation by phospholipids is inhibited by Ca²⁺-calmodulin. In the accompanying paper (Brzeska, H., Martin, B., and Korn, E. D. (1996) J. Biol. Chem. 271, 27049-27055), we identified the catalytic domain as the COOH-terminal 35 kDa produced by trypsin digestion of phosphorylated MIHC kinase. In this paper, we report the cloning and sequencing of the corresponding cDNA and expression of fully active catalytic domain. The expressed catalytic domain has substrate specificity similar to that of native kinase and resistance to trypsin similar to that of fully phosphorylated MIHC kinase. MIHC kinase catalytic domain has only 25% sequence identity to the catalytic domain of protein kinase A and similarly low sequence identity to the catalytic domains of protein kinase C- and calmodulin-dependent kinases, but 50% sequence identity and 70% similarity to the p21-activated kinase (PAK) and STE20 family of kinases. This suggests that MIHC kinase is (at least) evolutionarily related to the PAK family, whose activities are regulated by small GTP-binding proteins. The homology includes the presence of a potential MIHC kinase autophosphorylation site as well as conserved Tyr and Ser/Thr residues in the region corresponding to the P+1 loop of protein kinase A. A synthetic peptide corresponding to this region of MIHC kinase is phosphorylated by both the expressed catalytic domain and native MIHC kinase.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U67056[GenBank].
↵¹ The abbreviations used are:

MIHC kinase

myosin I heavy chain kinase

PAK

p21-activated kinase

PCR

polymerase chain reaction

PAGE

polyacrylamide gel electrophoresis

bp

base pair(s)

kb

kilobase pair(s)

Ni-NTA

nickel-nitrilotriacetic acid.
↵² Accession number: SWISS-PROT P35465; GenBank™ U23443[GenBank].
↵³ Accession number: SWISS-PROT Q03497[GenBank]; GenBank™ M94719[GenBank].
↵⁴ MIHC kinase phosphorylates Thr-18 in smooth muscle myosin II which is adjacent to Ser-19 which is phosphorylated by Ca²⁺-calmodulin-dependent myosin light chain kinase (Brzeska, H., Sellers, J. R., and Korn, E. D., unpublished data).
- Received July 3, 1996.
- Revision received August 16, 1996.