Abstract
The Dictyostelium myosin I heavy chain kinase (MIHCK) is a member of the p21-activated kinase family (Lee, S.-F., Egelhoff, T. T., Mahasneh, A., and Côté, G. P. (1996) J. Biol. Chem. 271, 27044-27048). MIHCK incubated with MgATP in the absence of effectors incorporates 1 mol of phosphate/mol, resulting in an approximately 40-fold increase in kinase activity. Sequence analysis of tryptic peptides has identified the major site of phosphorylation as Ser-8. A peptide and a glutathione S-transferase fusion protein containing the Ser-8 phosphorylation site were good substrates for MIHCK, indicating that MIHCK can catalyze its own activation. Guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)-Rac1 stimulates MIHCK autophosphorylation and kinase activity 10-fold. Phosphatidylserine, phosphatidylinositol, and phosphatidylinositol 4,5-bisphosphate, but not phosphatidylcholine or sphingosine, were as effective as GTPgammaS-Rac1 in enhancing MIHCK autophosphorylation and activity. Acidic lipids and GTPgammaS-Rac1 induced the autophosphorylation of a similar set of sites as judged by two-dimensional tryptic peptide maps. It is proposed that GTP-Rac and acidic phospholipids function cooperatively to associate MIHCK with membranes. Ca2+-calmodulin bound MIHCK and inhibited activation by acidic phospholipids but not by GTPgammaS-Rac1. These studies reveal a number of similarities between the regulatory properties of the Dictyostelium and Acanthamoeba MIHCK, suggesting that the signaling pathways that control myosin I are conserved.
Highlights
The Dictyostelium myosin I heavy chain kinase (MIHCK) is a member of the p21-activated kinase family
This step in the activation process occurs spontaneously in vitro when MIHCK is exposed to MgATP and is likely to be intermolecular in nature, because its rate is highly dependent on the MIHCK concentration [14]
Ser-8 was found to be a good substrate for MIHCK both in the context of a small synthetic peptide and within a glutathione S-transferase (GST) fusion protein containing the amino-terminal 42 residues of MIHCK
Summary
The Dictyostelium myosin I heavy chain kinase (MIHCK) is a member of the p21-activated kinase family Ca2؉calmodulin bound MIHCK and inhibited activation by acidic phospholipids but not by GTP␥S-Rac1 These studies reveal a number of similarities between the regulatory properties of the Dictyostelium and Acanthamoeba MIHCK, suggesting that the signaling pathways that control myosin I are conserved. Myosin I heavy chain kinases (MIHCKs) have been purified from Acanthamoeba and Dictyostelium and found by sequence analysis to have catalytic domains with a high degree of sequence identity to those of the PAK family kinases [15, 16]. The close relationship between MIHCK and other members of the PAK family is highlighted by the finding that Ste20p, Cla4p, and PAK efficiently phosphorylate and activate Acanthamoeba and Dictyostelium myosin I isoforms [17, 18], whereas Ste20p can phosphorylate the activating site in the head domain of yeast myosin I [13]
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