Calmodulin-binding and autoinhibitory domains of Acanthamoeba myosin I heavy chain kinase, a p21-activated kinase (PAK).
The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MIHCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated full-length MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca(2+)-calmodulin. In contrast to Dictyostelium MIHCK, however, Ca(2+)-calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.
Brzeska
H
H
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, MD 20892-8017, USA.
Young
R
R
Tan
C
C
Szczepanowska
J
J
Korn
E D
ED
D000048:Acanthamoeba / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry*; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007313:Insecta; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D024461:Myosin Type I / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D018076:DNA, Complementary; D010455:Peptides; D015800:Protozoan Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D024461:Myosin Type I; D008239:Lysine; D002118:Calcium