The Catalytic Domain of Acanthamoeba Myosin I Heavy Chain Kinase: II. EXPRESSION OF ACTIVE CATALYTIC DOMAIN AND SEQUENCE HOMOLOGY TO p21-ACTIVATED KINASE (PAK)

Hanna Brzeska,Joanna Szczepanowska,John Hoey,Edward D Korn

doi:10.1074/jbc.271.43.27056

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.

Highlights

Acanthamoeba MIHC1 kinase catalyzes the phosphorylation of a Ser or Thr residue in one of the actin-binding loops of the single heavy chains of the three Acanthamoeba myosin I isoenzymes [1] with a resultant 30-fold activation of their actinactivated Mg2ϩ-ATPase activities
Concluding Remarks— the linker region between invariant DFG and PE residues is not conserved among kinases, differing considerably in both length and secondary structure [12], the conformation and phosphorylation state of the linker region appear to be important for the activities of protein kinase A [15, 16, 18, 20], insulin receptor kinase [21], ERK2 kinase [22, 23], Cdk2 kinase [24, 25], and protein kinase C [17]
It is consistent with this proposal that the synthetic peptide (PK12) corresponding to this region is a good substrate for myosin I heavy chain (MIHC) kinase and the expressed catalytic domain (Table III)

Summary

Introduction

Acanthamoeba MIHC1 kinase catalyzes the phosphorylation of a Ser or Thr residue in one of the actin-binding loops of the single heavy chains of the three Acanthamoeba myosin I isoenzymes [1] with a resultant 30-fold activation of their actinactivated Mg2ϩ-ATPase activities (for summary, see Ref. 2). Kinase activity is activated by autophosphorylation of multiple sites and by Ca2ϩ-calmodulin-inhibited association with acidic phospholipids [3,4,5,6]; for details, see the Introduction in Ref. 7, and for review, see Ref. 8. The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) U67056. Provide additional evidence on the distribution of the multiple (up to 16) phosphorylation sites within the native kinase and show that the 35-kDa COOH-terminal fragment (F35) released by trypsin digestion has full catalytic activity and two to three of the phosphorylation sites of native kinase. We report the cloning and sequencing of the corresponding cDNA and expression of a fully active F35

Methods

Results

Conclusion