Myosin I heavy chain kinase: cloning of the full-length gene and acidic lipid-dependent activation by Rac and Cdc42.
Acanthamoeba myosin I heavy chain kinase (MIHCK) phosphorylates the heavy chains of amoeba myosins I, increasing their actin-activated ATPase activities. The activity of MIHCK is increased by binding to acidic phospholipids or membranes and by autophosphorylation at multiple sites. Phosphorylation at a single site is necessary and sufficient for full activation of the expressed catalytic domain. The rate of autophosphorylation of native MIHCK is controlled by a region N-terminal to the catalytic domain. By its substrate specificity and the sequence of its C-terminal catalytic domain, MIHCK was identified as a p21-activated kinase (PAK). We have now cloned the full-length genomic DNA and cDNA of MIHCK and have shown it to contain the conserved p21-binding site common to many members of the PAK family. Like some mammalian PAKs, MIHCK is activated by Rac and Cdc42, and this activation is GTP-dependent and accompanied by autophosphorylation. In contrast to mammalian PAKs, activation of MIHCK by Rac and Cdc42 requires the presence of acidic lipids. Also unlike mammalian PAK, MIHCK is not activated by sphingosine or other non-negatively charged lipids. The acidic lipid-binding site is near the N terminus followed by the p21-binding region. The N-terminal regulatory domain of MIHCK contains alternating strongly positive and strongly negative regions. and the extremely Pro-rich middle region of MIHCK has a strongly acidic N-terminal segment and a strongly basic C-terminal segment. We propose that autophosphorylation activates MIHCK by neutralizing the basic segment of the Pro-rich region, thus unfolding the regulatory domain and abolishing its inhibition of the catalytic domain.
Brzeska
H
H
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Young
R
R
Knaus
U
U
Korn
E D
ED
D000048:Acanthamoeba / Q000201:enzymology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008055:Lipids / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009218:Myosins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins
D008055:Lipids; D015800:Protozoan Proteins; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C033142:myosin-heavy-chain kinase; D019204:GTP-Binding Proteins; D009218:Myosins; D020744:rac GTP-Binding Proteins