Rajanala Kalpana, Sarkar Anshuk, Jhingan Gagan Deep, Priyadarshini Raina, Jalan Manisha, Sengupta Sagar, Nandicoori Vinay Kumar
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India
J Cell Sci. 2014 Aug 15;127(Pt 16):3505-20. doi: 10.1242/jcs.149112. Epub 2014 Jun 17.
A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions.
核孔复合体（NPC）核篮区域的主要成分核孔蛋白Tpr在调节多个重要过程中发挥作用。我们之前已经确定，Tpr以丝裂原活化蛋白激酶（MAP激酶）依赖性和MAP激酶非依赖性方式发生磷酸化，并且Tpr既是细胞外信号调节激酶2（也称为MAPK1）的底物，也是其支架。在此，我们报告在体内Tpr蛋白中鉴定出S2059和S2094作为主要的新型ERK非依赖性磷酸化位点，以及T1677、S2020、S2023和S2034作为额外的ERK非依赖性磷酸化位点。我们的结果表明蛋白激酶A使S2094残基磷酸化，并且该位点在有丝分裂期间发生过度磷酸化。此外，我们发现CDK1使Tpr的S2059残基磷酸化，并且磷酸化形式在末期与染色质明显共定位。S2059磷酸化的缺失消除了Tpr与Mad1的相互作用，从而损害了Mad1和Mad2蛋白的定位，导致细胞周期缺陷。Tpr上新的磷酸化位点的鉴定以及本研究中的观察结果有助于更好地理解Tpr的功能。
