Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation
Regulation of the polarity kinases PAR-1/MARK by 14-3-3 interaction and phosphorylation
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DOI：
10.1242/jcs.03097
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发表时间：
2006-10-01
影响因子：
4
通讯作者：
Alessi, Dario R.
中科院分区：
文献类型：
--
作者：
Goransson, Olga;Deak, Maria;Alessi, Dario R.
PAR-1/MARK激酶家族的成员在极性和细胞周期控制中发挥关键作用，并受14-3-3支架蛋白以及LKB 1肿瘤抑制激酶和非典型蛋白激酶C（PKC）的调节。在这项研究中，我们初步研究了哺乳动物MARK 3与14-3-3相互作用的机制。我们证明14-3-3与MARK 3的结合依赖于磷酸化，并且需要14-3-3的磷酸结合口袋。我们发现，与14-3-3的相互作用不是由先前表征的MARK 3磷酸化位点介导的，这使我们确定了15个新的磷酸化位点。这些位点的单点突变，以及先前鉴定的LKB 1-（T211）和非典型PKC位点（T564/S619），不破坏14-3-3结合。然而，其中所有17个磷酸化位点已转化为丙氨酸残基的突变体（称为17 A-MARK 3）不再能够结合14-3-3。野生型MARK 3存在于细胞质和质膜两者中，而17 A-MARK 3突变体显著地定位于质膜。我们提供的数据表明，MARK 3的膜定位需要一个高度保守的C-末端结构域，这被称为激酶相关结构域-1（KA-1）。我们还表明，14-3-3从MARK 3的解离不影响催化活性，并且MARK 3突变体，不能与14-3-3相互作用，是正常活性的。最后，我们建立了MARK亚型的亚细胞定位，以及非典型PKC过表达对14-3-3结合和定位的影响有显着差异。总的来说，这些结果表明14-3-3与MARK同种型的结合是由多个磷酸化位点介导的，并且用于在细胞质中锚MARK同种型。
Members of the PAR-1/MARK kinase family play critical roles in polarity and cell cycle control and are regulated by 14-3-3 scaffolding proteins, as well as the LKB1 tumour suppressor kinase and atypical protein kinase C (PKC). In this study, we initially investigated the mechanism underlying the interaction of mammalian MARK3 with 14-3-3. We demonstrate that 14-3-3 binding to MARK3 is dependent on phosphorylation, and necessitates the phosphate-binding pocket of 14-3-3. We found that interaction with 14-3-3 was not mediated by the previously characterised MARK3 phosphorylation sites, which led us to identify 15 novel sites of phosphorylation. Single point mutation of these sites, as well as the previously identified LKB1- (T211) and the atypical PKC sites (T564/S619), did not disrupt 14-3-3 binding. However, a mutant in which all 17 phosphorylation sites had been converted to alanine residues ( termed 17A-MARK3), was no longer able to bind 14-3-3. Wild-type MARK3 was present in both the cytoplasm and plasma membrane, whereas the 17A-MARK3 mutant was strikingly localised at the plasma membrane. We provide data indicating that the membrane localisation of MARK3 required a highly conserved C-terminal domain, which has been termed kinase-associated domain-1 (KA-1). We also show that dissociation of 14-3-3 from MARK3 did not affect catalytic activity, and that a MARK3 mutant, which could not interact with 14-3-3, was normally active. Finally, we establish that there are significant differences in the subcellular localisation of MARK isoforms, as well as in the impact that atypical PKC overexpression has on 14-3-3 binding and localisation. Collectively, these results indicate that 14-3-3 binding to MARK isoforms is mediated by multiple phosphorylation sites, and serves to anchor MARK isoforms in the cytoplasm.