The catalytic domain of Acanthamoeba myosin I heavy chain kinase (MIHCK) has been localized to the C-terminal 35-kDa. The catalytic domain contains 2-3 of the 7-8 autophosphorylation sites of the native 97-kDa protein with other sites in the middle third. Catalytic activity is lost when the 35-kDa domain is cleaved by trypsin into 25- and 10-kDa peptides. The catalytic domain has been cloned, sequenced and expressed in baculovirus-transfected insect cells. The amino acid sequence is 50% identical and 70% similar to mammalian PAK and yeast STE20, another member of the PAK family, which is activated by small G-proteins. The sequence similarity includes a potential MIHCK phosphorylation site in the region corresponding to the P+1 loop of PKA. The expressed catalytic domain is fully active and phosphorylated; it is inactivated by phosphatase-treatment and reactivated when autophosphorylated by incubation with ATP. About 14-kDa light chain (LC) of Acanthamoeba myosin IC (MIC) has been sequenced at the cDNA and protein level; it has greater sequence similarity to calmodulins than to any other myosin light chains in the data banks and 2 potential Ca2+-binding sites. The cDNA of the MIC heavy chain (HC) has also been cloned and MIC with fully active, phosphorylation-dependent actin-activated Mg2+-ATPase activity obtained by co-expression of the HC and LC cDNAs in baculovirus-infected insect cells. The cDNA corresponding to the coiled-coil alpha-helical rod of Acanthamoeba myosin II and mutant rods with the hinge region completely deleted or the Pro in the hinge region replaced by Ala have been expressed in and purified from E. coli. Electric birefringence studies show that the hinge of the expressed wild-type rod had the same bend- angle and flexibility as native enzyme while the deletion mutant was completely inflexible and the point mutant was not bent but did retain flexibility. Circular dichroism and differential scanning calorimetry showed highly cooperative, completely reversible thermal unfolding of the wild-type rod coupled to unfolding of the two chains. The data are consistent with a simple, two-state mechanism in which the dimeric rod unfolds as 2 unfolded monomers.
Showing the most recent 10 out of 21 publications
