Protein Kinase A Rescues Microtubule Affinity-regulating Kinase 2-induced Microtubule Instability and Neurite Disruption by Phosphorylating Serine 409

Si-Si Deng,Le-Yu Wu,Ya-Chao Wang,Peng-Rong Cao,Lei Xu,Qian-Ru Li,Meng Liu,Lun Zhang,Yue-Jing Jiang,Xiao-Yu Yang,Sheng-Nan Sun,Min-Jia Tan,Min Qian,Yi Zang,Linyin Feng,Jia Li

doi:10.1074/jbc.m114.629873

Abstract

Microtubule affinity-regulating kinase 2 (MARK2)/PAR-1b and protein kinase A (PKA) are both involved in the regulation of microtubule stability and neurite outgrowth, but whether a direct cross-talk exists between them remains unclear. Here, we found the disruption of microtubule and neurite outgrowth induced by MARK2 overexpression was blocked by active PKA. The interaction between PKA and MARK2 was confirmed by coimmunoprecipitation and immunocytochemistry both in vitro and in vivo. PKA was found to inhibit MARK2 kinase activity by phosphorylating a novel site, serine 409. PKA could not reverse the microtubule disruption effect induced by a serine 409 to alanine (Ala) mutant of MARK2 (MARK2 S409A). In contrast, mutation of MARK2 serine 409 to glutamic acid (Glu) (MARK2 S409E) did not affect microtubule stability and neurite outgrowth. We propose that PKA functions as an upstream inhibitor of MARK2 in regulating microtubule stability and neurite outgrowth by directly interacting and phosphorylating MARK2.

Highlights

Microtubule affinity-regulating kinase 2 (MARK2) and protein kinase A (PKA) are both involved in the regulation of microtubule stability
HEK293 cells were fixed and stained with antibodies against ␣-tubulin to visualize microtubules. pEGFP-MARK2 WT caused microtubule disruption and cell shrinkage in 60% of the transfected cell (Fig. 1, A, e-h, and C), which is extremely significant in contrast with the extended shape and intact microtubule network observed in the control cells (Fig. 1, A, a– d, and C) (p Ͻ 0.001) as reported previously [14]
When HEK293 cells were cotransfected with HA-PKAc (PKA catalytic subunit with an HA-YPYDVPDYA tag at its N terminus) and EGFP-MARK2 WT, the shrinking cells with disrupted microtubule significantly decreased by 41% compared with the cells transfected only with MARK2 WT (p ϭ 0.003) (Fig. 1, A, i–l, and C), whereas the overexpression of PKAc did not influence the protein amount of the transfected EGFP-MARK (Fig. 1B)

Summary

Introduction

MARK2 and PKA are both involved in the regulation of microtubule stability. Results: PKA significantly inhibits MARK2 by phosphorylating serine 409 and rescues MARK2-induced microtubule instability and neurite disruption. Conclusion: PKA functions as an upstream inhibitor of MARK2 by directly interacting and phosphorylating it. We found the disruption of microtubule and neurite outgrowth induced by MARK2 overexpression was blocked by active PKA. PKA was found to inhibit MARK2 kinase activity by phosphorylating a novel site, serine 409. PKA could not reverse the microtubule disruption effect induced by a serine 409 to alanine (Ala) mutant of MARK2 (MARK2 S409A). We propose that PKA functions as an upstream inhibitor of MARK2 in regulating microtubule stability and neurite outgrowth by directly interacting and phosphorylating MARK2

Methods

Results

Conclusion