van der Laden Jan, Soppa Ulf, Becker Walter
Institute of Pharmacology and Toxicology, RWTH Aachen University, Wendlingweg 2, 52057, Aachen, Germany.
Cell Commun Signal. 2015 Jan 29;13:3. doi: 10.1186/s12964-014-0082-6.
Homeodomain interacting protein kinases (HIPKs) function as modulators of cellular stress responses and regulate cell differentiation, proliferation and apoptosis. The HIPK family includes HIPK1, HIPK2 and HIPK3, which share a similar domain structure, and the more distantly related HIPK4. Although HIPKs phosphorylate their substrates on serine or threonine residues, it was recently reported that HIPK2 depends on the autophosphorylation of a conserved tyrosine in the activation loop to acquire full catalytic activity and correct subcellular localization. In this study we addressed the question whether tyrosine autophosphorylation in the activation loop has a similar function in the other members of the HIPK family.
All HIPKs contained phosphotyrosine when expressed in HeLa cells. Catalytically inactive point mutants were not tyrosine-phosphorylated, indicating that HIPKs are dual-specificity protein kinases that autophosphorylate on tyrosine residues. HIPK point mutants lacking the conserved tyrosine residue in the activation loop showed reduced catalytic activity towards peptide and protein substrates. Analysis of these mutants revealed that HIPK1, HIPK2 and HIPK3 but not HIPK4 are capable of autophosphorylating on other tyrosines. Inhibition of tyrosine phosphatase activity by treatment with vanadate enhanced global phosphotyrosine content of HIPK1, HIPK2 and HIPK3 but did not affect tyrosine phosphorylation in the activation loop. Mutation of the activation-loop tyrosines resulted in a redistribution of HIPK1 and HIPK2 from a speckle-like subnuclear compartment to the cytoplasm, whereas catalytically inactive point mutants showed the same pattern of cellular distribution as the wild type proteins. In contrast, mutation of the activating tyrosine did not increase the low percentage of cells with extranuclear HIPK3. HIPK4 was excluded from the nucleus with no difference between the wild type kinase and the point mutants.
These results show that HIPKs share the mechanism of activation by tyrosine autophosphorylation with the closely related DYRK family (dual-specificity tyrosine phosphorylation regulated kinase). However, members of the HIPK family differ regarding the subcellular localization and its dependence on tyrosine autophosphorylation.
同源结构域相互作用蛋白激酶（HIPKs）作为细胞应激反应的调节因子，可调控细胞分化、增殖和凋亡。HIPK家族包括结构域相似的HIPK1、HIPK2和HIPK3，以及亲缘关系较远的HIPK4。尽管HIPKs可使丝氨酸或苏氨酸残基上的底物磷酸化，但最近有报道称，HIPK2依赖于激活环中保守酪氨酸的自身磷酸化来获得完整的催化活性和正确的亚细胞定位。在本研究中，我们探讨了激活环中的酪氨酸自身磷酸化在HIPK家族其他成员中是否具有类似功能。
当在HeLa细胞中表达时，所有HIPKs均含有磷酸酪氨酸。催化无活性的点突变体未发生酪氨酸磷酸化，表明HIPKs是双特异性蛋白激酶，可在酪氨酸残基上自身磷酸化。激活环中缺乏保守酪氨酸残基的HIPK点突变体对肽和蛋白质底物的催化活性降低。对这些突变体的分析表明，HIPK1、HIPK2和HIPK3能够在其他酪氨酸上自身磷酸化，但HIPK4不能。用钒酸盐处理抑制酪氨酸磷酸酶活性可增加HIPK1、HIPK2和HIPK3的整体磷酸酪氨酸含量，但不影响激活环中的酪氨酸磷酸化。激活环酪氨酸的突变导致HIPK1和HIPK2从斑点状核内区室重新分布到细胞质中，而催化无活性的点突变体显示出与野生型蛋白相同的细胞分布模式。相比之下，激活酪氨酸的突变并未增加核外HIPK3细胞的低比例。HIPK4被排除在细胞核外，野生型激酶和点突变体之间无差异。
这些结果表明，HIPKs与密切相关的DYRK家族（双特异性酪氨酸磷酸化调节激酶）共享通过酪氨酸自身磷酸化激活的机制。然而，HIPK家族成员在亚细胞定位及其对酪氨酸自身磷酸化的依赖性方面存在差异。
