ERK activation by Rab2B in the early secretory pathway impacts the ERGIC-Golgi interface

Abstract

The Golgi complex is a hub for several signal transduction networks that regulate Golgi morphology, membrane transport, and glycosylation. The Rab2 (A, B isoforms) protein participates in membrane trafficking to and from the Golgi and is also linked to signaling molecules. In that regard, Rab2A in breast cancer stem cells binds and blocks (p)ERK1/2 inactivation by MAP kinase phosphatase 3. However, the cellular role of Rab2B in ERK1/2 signaling activity at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC/IC) and cis Golgi where Rab2B immunolocalizes and functions is unknown. To address this question, normal rat kidney (NRK) cells were transfected with Rab2B cDNA to mimic Rab2 overexpression as found in cancer cells. Rab2B overexpressing NRK cells had a significant increase in steady state activated ERK. Studies were then performed to identify the Rab2-ERK1/2 substrate(s) that locate and function in the early secretory pathway. To that end, GRASP65 was identified as a target of ERK1/2 phosphorylation. In Rab2B overexpressing NRK cells, GRASP65 co-distributed with GM130 on membranes of the ERGIC/IC that increased in size and number with the concomitant appearance of unlinked cis Golgi elements. Additionally, we observed GRASP65 labeled ERGIC/IC membranes that accumulated at 15°C and remained prominent after temperature shift to 37 °C to promote transport. However, addition of a MEK inhibitor reversed the transport block indicating that ERK1/2 phosphorylation of GRASP65 effected ERGIC/IC redistribution to the cis Golgi. Since several glycosyltransferases cycle between the Golgi and ERGIC/IC, a potential consequence of Golgi structural changes is modification of protein glycosylation. Indeed, we found changes in total and cell surface O-glycosylation in Rab2B overexpressing cells. These results suggest that phosphoGRASP65 plays an important role in the protein sorting and recycling process from the ERGIC/IC to cis Golgi: Dysregulation results in cis Golgi discontinuities and aberrant glycosylated proteins that are potentially pro-oncogenic.