REGULATION OF ACANTHAMOEBA MYOSINS AND MYOSIN KINASES (PAKS)

肌球蛋白I的生物化学特征：网状曲霉的单一肌球蛋白I在其重链(MyoA)的TEDS位含有一个Ser残基。MyoA缺失突变体的致死性可以通过表达野生型重链或突变体来逆转，在这些突变体中，这个Ser被Ala或Glu取代。因此，这种肌球蛋白要么是肌球蛋白的肌动蛋白依赖的镁ATPase活性需要TEDS位点的Glu、Asp或磷酸化的丝氨酸或苏氨酸的例外，要么是肌球蛋白I的基本功能(S)不依赖于其催化活性。我们现在已经用野生型重链或突变重链(其中Glu或Ala取代了TEDS位点上的SerSer)与曲霉钙调蛋白(曲霉肌球蛋白I的轻链)进行了共表达。野生型的肌动蛋白依赖的镁ATPase活性是Glu突变体3/S的10/S，而ala突变体没有肌动蛋白依赖的镁ATPase活性或活性很低(三种酶都有相同的K/EDTA-ATPase活性)。因此，肌球蛋白I的基本功能(S)似乎几乎不需要肌动蛋白依赖的镁ATPase活性。正在进行的研究表明，野生型肌球蛋白中的TEDS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的镁ATPase活性。PAK家族成员棘阿米巴肌球蛋白I重链激酶(MIHCK)调控区域的定位：只有少数PAK家族成员在蛋白质水平上被鉴定。MIHCK是一个79 kDa的蛋白质，具有一个35 kDa的C端类似PAK的催化结构域，在所有PAK中都是保守的，以及一个p21结合结构域，在大多数PAK中存在于其调控的N末端区域。在MIHCK和其他PAK之间没有其他序列同源性区域。MIHCK活性通过多个位点的自动磷酸化而上调，其中包括催化区域中的一个。就像哺乳动物的α-、β-和伽马-PAK一样。脂质和RAC或CdC42刺激MIHCK的自磷酸化。与哺乳动物的Pak不同，MIHCK的自动磷酸化需要酸性脂类，而p21只有在酸性脂类存在的情况下才能激活。钙调素与MIHCK结合，消除脂质活化。我们先前提出，MIHCK通过其N端抑制区和C端催化结构域的相互作用而维持在其非活性状态。通过表达MIHCK片段，我们现在已经将自抑制区映射到p21结合部位，残基91-157，并将钙调蛋白结合部位映射到之前的碱基区，残基51-79。含有残基51-157的片段抑制天然的非磷酸化的MIHCK的活性，但对自动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了自动磷酸化。棘阿米巴肌球蛋白I重链激酶催化结构域(MIHCK)转导HeLa细胞和NIH3T3细胞对局部ADHESIION复合体的影响：我们用GFP偶联的MIHCK催化结构域瞬时转染HeLa和NIH3T3细胞，MIHCK是一种活性远高于哺乳动物PAK的PAK。荧光显微镜下，肌动蛋白和肌球蛋白II在细胞内的分布无明显变化。然而，根据纽蛋白、他林和帕西林抗体的染色结果，局灶性粘连的分布受到显著影响。转染活性激酶的细胞的黏附复合体主要分布在细胞的外围，而两个催化失活激酶突变体或单独转染GFP的细胞中黏附复合体分布均匀。与这些观察结果一致的是，用活性的MIHCK催化结构域转染细胞显著增加了与底物失去接触的细胞的比例。分离的细胞既不坏死，也不凋亡，大小与对照细胞相同，但形状不同。将哺乳动物PAK催化结构域导入HeLa细胞后，细胞表型无明显变化。这些观察结果表明，MIHCK催化结构域对黏附复合体的重新分布与其高催化活性有关。我们现在正试图确定MIHCK的底物可能对这些影响负责。棘阿米巴肌球蛋白II棒替代棘阿米巴肌球蛋白II棒的效果：棘阿米巴肌球蛋白II的肌动蛋白依赖的镁ATPase活性受杆端三个丝氨酸残基的磷酸化负调控。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型肌球蛋白重链，其中Dictyostelialmyosin II头与野生型棘阿米巴肌球蛋白II杆或突变杆融合，三个Ser残基被天冬氨酸或丙氨酸取代。嵌合肌球蛋白在Dictyostelialmyosin II缺失细胞中表达，该细胞既不能在悬浮培养中分裂，也不能在固体基质上分化为子实体。在表达Dictyostelialmyosin II头和骨骼肌球蛋白杆状嵌合体的细胞中，这三种嵌合肌球蛋白的细胞在悬浮培养中生长和转化DictyostelialWild型肌球蛋白II的细胞一样。这三种嵌合肌球蛋白的肌动蛋白依赖的镁ATPase活性比野生型Dictyostelialmyosin II高约10倍，并且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的棘阿米巴棒的磷酸化并不抑制其肌动蛋白依赖的镁ATPase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.