Tan I, Ng C H, Lim L, Leung T
Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore and Institute of Neurology, University College London, London WC1N 1PJ, United Kingdom.
J Biol Chem. 2001 Jun 15;276(24):21209-16. doi: 10.1074/jbc.M102615200. Epub 2001 Apr 3.
The myotonic dystrophy kinase-related kinases RhoA binding kinase and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, for actin reorganization. Using substrate screening in various tissues, we uncovered two major substrates, p130 and p85, for MRCKalpha-kinase. p130 is identified as myosin binding subunit p130, whereas p85 is a novel related protein. p85 contains N-terminal ankyrin repeats, an alpha-helical C terminus with leucine repeats, and a centrally located conserved motif with the MRCKalpha-kinase phosphorylation site. Like MBS130, p85 is specifically associated with protein phosphatase 1delta (PP1delta), and this requires the N terminus, including the ankyrin repeats. This association is required for the regulation of both the catalytic activities and the assembly of actin cytoskeleton. The N terminus, in association with PP1delta, is essential for actin depolymerization, whereas the C terminus antagonizes this action. The C-terminal effects consist of two independent events that involved both the conserved phosphorylation inhibitory motif and the alpha-helical leucine repeats. The former was able to interact with PP1delta only in the phosphorylated state and result in inactivation of PP1delta activity. This provides further evidence that phosphorylation of a myosin binding subunit protein by specific kinases confers conformational changes in a highly conserved region that plays an essential role in the regulation of its catalytic subunit activities.
强直性肌营养不良激酶相关激酶RhoA结合激酶和强直性肌营养不良激酶相关Cdc42结合激酶（MRCK）分别是RhoA和Cdc42用于肌动蛋白重组的效应器。通过在各种组织中进行底物筛选，我们发现了MRCKα激酶的两种主要底物，即p130和p85。p130被鉴定为肌球蛋白结合亚基p130，而p85是一种新的相关蛋白。p85包含N端锚蛋白重复序列、带有亮氨酸重复序列的α螺旋C端以及位于中央的带有MRCKα激酶磷酸化位点的保守基序。与MBS130一样，p85与蛋白磷酸酶1δ（PP1δ）特异性结合，这需要包括锚蛋白重复序列在内的N端。这种结合对于肌动蛋白细胞骨架的催化活性和组装的调节都是必需的。与PP1δ结合的N端对于肌动蛋白解聚至关重要，而C端则拮抗这种作用。C端的作用由两个独立事件组成，涉及保守的磷酸化抑制基序和α螺旋亮氨酸重复序列。前者仅在磷酸化状态下能够与PP1δ相互作用并导致PP1δ活性失活。这进一步证明了特定激酶对肌球蛋白结合亚基蛋白的磷酸化在一个高度保守区域赋予构象变化，该区域在其催化亚基活性的调节中起关键作用。
