BIOCHEMMICAL CHARACTERIZATION OF ASPERGILLUS MYOSIN I: The single myosin I of Aspergillus nidulans contains a Ser residue at the TEDS-site of its heavy chain (myoA). The lethality of the myoA null mutant is reversed by expression of wildtype heavy chain or mutants in which this Ser is replaced by Ala or Glu. Therefore, either this myosin is an exception to the rule that the actin-dependent MgATPase activity of myosins requires Glu, Asp or phosphorylated Ser or Thr at the TEDS-site or the essential function(s)of Aspergillus myosin I do not depend on its catalytic activity. We have now co-expressed Aspergillus calmodulin (the light chain of Aspergillus myosin I) with either wildtype heavy chain or mutant heavy chains in which Glu or Ala replace Ser at the TEDS-site. The actin-dependent MgATPase activity of the wildtype was 10/s, of the Glu-mutant 3/s, and the Ala mutant had no, or very low, actin-dependent MgATPase activity (all three enzymes had equal K/EDTA-ATPase activity). Thus, the essential function(s) of Aspergillus myosin I seem to require little, if any, actin-dependent MgATPase activity. Research in progress suggests that the TEDS-site Ser in the wildtype myosin is phosphorylated during expression which would explain its very high actin-dependent MgATPase activity. LOCALIZATION OF THE REGULATORY REGIONS OF ACANTHAMOEBA MYOSIN I HEAVY CHAIN KINASE (MIHCK), A MEMBER OF THE PAK FAMILY: Only a few members of the growing PAK family have been characterized at the protein level. MIHCK is a 79-kDa protein with a 35-kDa C-terminal PAK-like catalytic domain, that is conserved in all PAKs, and a p21-binding domain, that is present in most PAKs, within its regulatory N-terminal region. There are no other regions of sequence homology between MIHCK and other PAKs. MIHCK activity is up-regulated by autophosphorylation of multiple sites including one in the catalytic domain. Like mammalian alpha-, beta- and gamma-PAK. autophosphorylation of MIHCK is stimulated by lipids and Rac or Cdc42. Unlike mammalian PAKs, autophosphorylation of MIHCK requires acidic lipids, and p21s activate only in the presence of acidic lipids. Ca-calmodulin binds to MIHCK and eliminates lipid activation. We had previously proposed that MIHCK is maintained in its inactive state by interaction of its N-terminal inhibitory region with its C-terminal catalytic domain. By expressing fragments of MIHCK, we have now mapped the autoinhibitory region to the p21-binding site, residues 91-157, and mapped the calmodulin-binding site to the preceding basic region, residues 51-79. Fragments that contain residues 51-157 inhibit the activity of native, unphosphorylated MIHCK but have no effect on the activity of autophosphorylated, fully active MIHCK, suggesting that these fragments inhibit autophosphorylation. TRANSFECTION OF HELA CELLS AND NIH 3T3 CELLS BY THE CATALYTIC DOMAIN OF ACANTHAMOEBA MYOSIN I HEAVY CHAIN KINASE (MIHCK) AFFECTS FOCAL ADHESIION COMPLEXES: We transiently transfected HeLa and NIH 3T3 cells with GFP-coupled catalytic domain of MIHCK, a PAK whose activity is much higher than that reported for mammalian PAKs. By fluorescence microscopy, there were no significant changes in the distribution of actin or myosin II in the transfected cells. However, as determined by staining with antibodies against vinculin, talin and paxillin, the distribution of focal adhesions was significantly affected. Adhesion complexes of cells transfected with active kinase were localized mostly in the cell periphery whereas adhesion complexes were uniformly distributed in cells transfected with two catalytically inactive kinase mutants or GFP alone. Consistent with these observations, transfection with active MIHCK catalytic domain substantially increased the fraction of cells that lost contact with the substratum. The detached cells were neither necrotic nor apoptotic and were the same size as control cells, but with different shape. Transfection of HeLa cells with the catalytic domain of mammalian PAK caused no substantial change in phenotype. These observations suggest that the redistribution of adhesion complexes by the MIHCK catalytic domain are associated with its high catalytic activity. We are now attempting to identify the substrates of MIHCK that may be responsible for these effects. EFFECT OF SUBSTITUTION OF THE ACANTHAMOEBA MYOSIN II ROD FOR THE DICTYOSTELIUM MYOSIN II ROD: The actin-dependent MgATPase activity of Acanthamoeba myosin II is negatively regulated by phosphorylation of three serine residues at the end of rod. To investigate the structural basis of the communication between the N-terminal head and C-terminal tail, we constructed three chimeric myosin heavy chains in which the Dictyostelium myosin II head was fused with either wild type Acanthamoeba myosin II rod or mutant rods with the three Ser residues replaced by either Asp or Ala. The chimeric myosins were expressed in Dictyostelium myosin II null cells that neither divide in suspension culture nor differentiate to fruiting bodies on solid substratum. As shown previously for cells expressing a chimera of Dictyostelium myosin II head and skeletal myosin rod, cells transfected with the three chimeric myosins grew in suspension culture as well as cells transformed with Dictyostelium wildtype myosin II. The actin-dependant MgATPase activities of the three chimeric myosins were about 10-fold higher than wildtype Dictyostelium myosin II and only slightly activated by regulatory light chain phosphorylation. Experiments in progress indicate that phosphorylation of the Acanthamoeba rod of the wildtype chimeric myosin II does not inhibit its actin-dependent MgATPase activity.
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