Convergence of Cell Cycle Regulation and Growth Factor Signals on GRASP65

Shin-Ichiro Yoshimura,Katsuji Yoshioka,Francis A Barr,Martin Lowe,Kazuhisa Nakayama,Shoji Ohkuma,Nobuhiro Nakamura

doi:10.1074/jbc.m502442200

Abstract

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. During mitosis, GRASP65 is heavily phosphorylated, and in turn, cisternal stacking is inhibited leading to the breakdown of the Golgi apparatus. Here we show that GRASP65 is phosphorylated on serine 277 in interphase cells, and this is strongly enhanced in response to the addition of serum or epidermal growth factor. This is directly mediated by ERK suggesting that GRASP65 has some role in growth factor signal transduction. Phosphorylation of Ser-277 is also dramatically increased during mitosis, however this is mediated by Cdk1 and not by ERK. The microinjection of recombinant GRASP65 without N-terminal myristoylation or a peptide fragment containing Ser-277 into the cytosol of normal rat kidney cells inhibits passage through mitosis. This effect is abolished when Ser-277 is replaced with alanine suggesting the phosphorylation of Ser-277 plays an important role in cell cycle regulation. The convergence of cell cycle regulation and growth factor signals on GRASP65 Ser-277 suggests that GRASP65 may function as a signal integrator controlling the cell growth.

Highlights

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells
It is probable that the one or more kinases responsible for the GRASP65 phosphorylation localize to the Golgi apparatus in untreated interphase cells, and this is segregated together with other Golgi proteins from GRASP65 after brefeldin A (BFA) treatment such that it can no longer efficiently phosphorylate GRASP65
We have confirmed that purified Cdk1-cyclin B can phosphorylate GRASP65.2 These results strongly suggested that the serine 277 (Ser-277) is phosphorylated by Cdk1-cyclin B under mitotic condition

Summary

Introduction

Together with other Golgi matrix components, GRASP65 contributes to the stacking of Golgi cisternae in interphase cells. MEK1,1 and not Cdk1-cyclin B, was shown to be responsible for the disassembly of the Golgi apparatus by in vitro reconstitution experiments using a permeabilized cell system [5]. GRASP65 (golgi reassembly stacking protein 65) is a peripheral membrane protein enriched on the cis-face of the Golgi apparatus It was identified as an N-ethylmaleimide-sensitive factor required for the stacking of Golgi cisternae from mitotically disassembled Golgi fragments in a cell-free system [7]. The Golgi cisternae have to be unstacked and fragmented after reaching the trans position, and their content delivered to the plasma membrane, endosomes, or regulated secretory granules It is still under debate which of these models or a mixed intermediate model is correct

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Conclusion