Schwaid Adam G, Su Chunyan, Loos Paula, Wu Jiang, Nguyen Chuong, Stone Kathryn L, Kanyo Jean, Geoghegan Kieran F, Bhattacharya Samit K, Dow Robert L, Buckbinder Leonard, Carpino Philip A
Structural Biology and Biophysics, Center for Chemistry Innovation and Excellence, Pfizer Pharmatherapeutics Research and Development , Groton, Connecticut 06340, United States.
W.M. Keck Foundation Biotechnology Resource Laboratory, Yale University , New Haven, Connecticut 06520, United States.
ACS Chem Biol. 2015 Dec 18;10(12):2667-71. doi: 10.1021/acschembio.5b00679. Epub 2015 Sep 30.
Mitogen-activated protein kinase 4 (MAP4K4) regulates the MEK kinase cascade and is implicated in cytoskeletal rearrangement and migration; however, identifying MAP4K4 substrates has remained a challenge. To ascertain MAP4K4-dependent phosphorylation events, we combined phosphoproteomic studies of MAP4K4 inhibition with in vitro assessment of its kinase specificity. We identified 235 phosphosites affected by MAP4K4 inhibition in cells and found that pTP and pSP motifs were predominant among them. In contrast, in vitro assessment of kinase specificity showed that MAP4K4 favors a pTL motif. We showed that MAP4K4 directly phosphorylates and coimmunoprecipitates with FERM, RhoGEF, and pleckstrin domain-containing protein 1 (FARP1). MAP4K4 inhibition in SH-SY5Y cells increases neurite outgrowth, a process known to involve FARP1. As FARP1 and MAP4K4 both contribute to cytoskeletal rearrangement, the results suggest that MAP4K4 exerts some of its effects on the cytoskeleton via phosphorylation of FARP1.
丝裂原活化蛋白激酶4（MAP4K4）调节MEK激酶级联反应，并参与细胞骨架重排和迁移；然而，鉴定MAP4K4的底物仍然是一项挑战。为了确定依赖MAP4K4的磷酸化事件，我们将MAP4K4抑制的磷酸蛋白质组学研究与对其激酶特异性的体外评估相结合。我们在细胞中鉴定出235个受MAP4K4抑制影响的磷酸化位点，发现其中pTP和pSP基序占主导。相比之下，激酶特异性的体外评估表明，MAP4K4倾向于pTL基序。我们发现MAP4K4直接磷酸化含FERM、RhoGEF和普列克底物蛋白结构域蛋白1（FARP1）并与之共免疫沉淀。在SH-SY5Y细胞中抑制MAP4K4可增加神经突生长，这一过程已知涉及FARP1。由于FARP1和MAP4K4都参与细胞骨架重排，结果表明MAP4K4通过磷酸化FARP1对细胞骨架发挥某些作用。
