REGULATION OF ACANTHAMOEBA MYOSINS AND MYOSIN KINASES (PAKS)

曲霉MYOSIN I的生化特性：细粒曲霉（ASPERGILLUS nidulans）的单个MYOSIN I在其重链（myoA）的ted位点上含有一个丝氨酸残基。myoA零突变体的致死率可以通过表达野生型重链或突变体来逆转，其中Ser被Ala或Glu取代。因此，这个肌凝蛋白是肌凝蛋白的肌动蛋白依赖MgATPase活性需要在ted -位点上的Glu， Asp或磷酸化的Ser或Thr的规则的例外，或者曲霉肌凝蛋白I的基本功能不依赖于它的催化活性。我们现在已经将曲霉钙调蛋白（曲霉肌球蛋白I的轻链）与野生型重链或突变型重链共同表达，其中Glu或Ala取代了tds位点的Ser。野生型的肌动蛋白依赖性MgATPase活性为10/s， glu突变体为3/s， Ala突变体没有或非常低的肌动蛋白依赖性MgATPase活性（所有三种酶的K/ edta - atp酶活性相等）。因此，曲霉肌球蛋白I的基本功能似乎只需要很少（如果有的话）依赖于MgATPase的活性。正在进行的研究表明，野生型肌球蛋白的ted位点Ser在表达过程中被磷酸化，这可以解释其非常高的肌动蛋白依赖性MgATPase活性。棘阿米巴MYOSIN I重链激酶（MIHCK）调控区域的定位，PAK家族成员之一：只有少数正在生长的PAK家族成员在蛋白质水平上被表征。MIHCK是一种79-kDa的蛋白，具有35-kDa的c端pak样催化结构域，在所有PAKs中都是保守的，并且在其调节的n端区域内存在于大多数PAKs中p21结合结构域。MIHCK与其他PAKs之间没有其他序列同源区域。MIHCK活性通过多个位点的自磷酸化而上调，包括催化区域的一个位点。就像哺乳动物的-，-和- pak。脂质和Rac或Cdc42刺激MIHCK的自磷酸化。与哺乳动物PAKs不同，MIHCK的自磷酸化需要酸性脂质，而p21s仅在酸性脂质存在时激活。钙钙调素与MIHCK结合，消除脂质活化。我们之前提出，MIHCK是通过其n端抑制区域与c端催化区域的相互作用维持其非活性状态的。通过表达MIHCK片段，我们现在已经将自身抑制区域定位到p21结合位点，残基91-157，并将钙调素结合位点定位到先前的基本区域，残基51-79。含有残基51-157的片段抑制天然未磷酸化的MIHCK的活性，但对自磷酸化的完全活性的MIHCK的活性没有影响，这表明这些片段抑制自磷酸化。棘阿米巴肌球蛋白I重链激酶（MIHCK）催化结构域转染HELA细胞和NIH 3T3细胞影响局灶黏附复合物：我们用gfp偶联的MIHCK催化结构域瞬时转染HELA细胞和NIH 3T3细胞，MIHCK的活性远高于哺乳动物的PAKs。荧光显微镜下，转染细胞内肌动蛋白和肌球蛋白II的分布未见明显变化。然而，通过抗vinculin、talin和paxillin的抗体染色，发现局灶性粘连的分布受到了显著影响。转染活性激酶的细胞的粘附复合物主要定位于细胞周围，而转染两种催化失活激酶突变体或单独转染GFP的细胞的粘附复合物则均匀分布。与这些观察结果一致，转染活性MIHCK催化结构域大大增加了与基质失去接触的细胞比例。离体细胞既不坏死也不凋亡，大小与对照细胞相同，但形状不同。用哺乳动物PAK催化结构域转染HeLa细胞后，其表型未发生实质性变化。这些观察结果表明，通过MIHCK催化结构域重新分配粘附配合物与其高催化活性有关。我们现在正试图确定可能导致这些影响的MIHCK底物。棘阿米巴肌球蛋白II棒替代dictyostium MYOSIN II棒的作用：棘阿米巴肌球蛋白II的肌动蛋白依赖性MgATPase活性受到棒末端三个丝氨酸残基磷酸化的负调控。为了研究n端头和c端尾之间通讯的结构基础，我们构建了三条嵌合肌球蛋白重链，其中Dictyostelium myosin II头与野生型棘阿米巴myosin II棒或突变棒融合，其中三个Ser残基被Asp或Ala取代。嵌合肌球蛋白在Dictyostelium myosin II无细胞中表达，该细胞在悬浮培养中既不能分裂，也不能在固体基质上分化为子实体。如前所示，表达Dictyostelium myosin II头和骨肌球蛋白棒嵌合体的细胞，转染了三种嵌合肌球蛋白的细胞在悬浮培养中生长，以及用Dictyostelium野生型肌球蛋白II转化的细胞生长。三种嵌合肌球蛋白的肌动蛋白依赖性MgATPase活性比野生型盘基骨桥菌肌球蛋白II高约10倍，仅被调节轻链磷酸化轻微激活。正在进行的实验表明，野生型嵌合肌球蛋白II棘阿米巴棒的磷酸化不会抑制其肌动蛋白依赖性MgATPase活性。

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.