Mitotic Phosphorylation of Golgi Reassembly Stacking Protein 55 by Mitogen-activated Protein Kinase ERK2

Abstract

The role of the mitogen-activated protein kinase kinase (MKK)/extracellular-activated protein kinase (ERK) pathway in mitotic Golgi disassembly is controversial, in part because Golgilocalized targets have not been identified. We observed that Golgi reassembly stacking protein 55 (GRASP55) was phosphorylated in mitotic cells and extracts, generating a mitosis-specific phospho-epitope recognized by the MPM2 mAb. This phosphorylation was prevented by mutation of ERK consensus sites in GRASP55. GRASP55 mitotic phosphorylation was significantly reduced, both in vitro and in vivo, by treatment with U0126, a potent and specific inhibitor of MKK and thus ERK activation. Furthermore, ERK2 directly phosphorylated GRASP55 on the same residues that generated the MPM2 phospho-epitope. These results are the first demonstration of GRASP55 mitotic phosphorylation and indicate that the MKK/ERK pathway directly phosphorylates the Golgi during mitosis. INTRODUCTIONIn preparation for cell division, the highly ordered stacked cisternae of the mammalian Golgi complex undergo mitotic breakdown (Roth, 1999). This breakdown is triggered by protein phosphorylation events (Nelson, 2000) that function, at least in part, to inhibit one or more trafficking steps (Lowe et al., 1998b). The exact nature of the Golgi breakdown product is controversial in that it may comprise dispersed small vesicles (Jesch and Linstedt, 1998), clustered small vesicles (Shima et al., 1998), or membranes that fuse with the ER (Zaal et al., 1999). In any event, the disassembled Golgi is subsequently partitioned into each daughter cell where it reassembles a ribbon of stacked membranes positioned near the microtubule organizing center.Currently, the identity of the protein kinases that mediate Golgi disassembly is controversial. Both cyclin-dependent kinase 1 (CDK), considered a master regulatory kinase in the G2/M transition (Ohi and Gould, 1999), and a kinase in the mitogen-activated protein kinase (MAPK) signaling pathway known as MAPK kinase (MKK) have been implicated in mitotic Golgi breakdown. In its better characterized role in cell cycle entry from Go, MKK (present as 2 isoforms, MKK1 and MKK2), becomes activated by phosphorylation and then phosphorylates and activates the MAP kinase isoforms known as extracellular signal-regulated protein kinase (ERK) 1 and ERK2. Importantly, the MKK and ERK isoforms are present in their active forms in mitotic extracts (Kuang and Ashorn, 1993) and cells (Shapiro et al., 1998;Zecevic et al., 1998;Tolwinski et al., 1999), and mammalian fibroblasts treated with an MKK inhibitor arrest in G2 failing to enter M-phase (Wright et al., 1999). One of the first observations implicating MKK in mitosis came from an assay that measures mitotic cytosol-dependent Golgi disassembly in permeabilized normal rat kidney cells. In this assay the Golgi breakdown activity is separable from CDK1 and is blocked by MKK inactivation or MKK immunodepletion (Acharya et al., 1998). In contrast, in an assay that measures mitotic cytosol-dependent disas...

GRASP55 Regulates Golgi Ribbon Formation

“…We hypothesize that the mechanism involves MEK pathway phospho-inhibition of GRASP55-mediated Golgi linking. GRASP55 is a mitotic target of the MEK/ERK pathway (Jesch et al, 2001), and as shown above, a phospho-mimic version of GRASP55 fails to link the Golgi. Furthermore, a nonphosphorylatable version of GRASP55 blocks both G2 Golgi unlinking and normal G2/M kinetics (Feinstein and Linstedt, 2007).…”

“…Primary antibodies used were as follows: anti-Myc 9e10 (Jesch et al, 2001), anti-giantin (Linstedt and Hauri, 1993), anti-p115 (Puthenveedu and Linstedt, 2001), anti-GM130 (Puthenveedu and Linstedt, 2001), anti-GP73 (Bachert et al, 2007), anti-GPP130 (Linstedt et al, 1997, and anti-GRASP55, which was generated by immunizing rabbits (Covance, Emeryville, CA) with purified glutathione transferase (GST)-GRASP55 (Jesch et al, 2001). Affinity-purified, conjugated secondary antibodies were purchased (Invitrogen, Carlsbad, CA).…”

“…To test the influence of GRASP55 phosphorylation on Golgi linking, we substituted aspartic acids in place of two threonines known to be mitotically phosphorylated by ERK (Jesch et al, 2001). Gene replacement after knockdown was carried out as described …”

“…U0126 (used at 10 M) was from Promega (Madison, WI), thymidine (used at 2 mM) was from EMD Biosciences (Darmstadt, Germany), and nocodazole (used at 500 nM) and olomoucine II (used at 10 M) were from Sigma-Aldrich (St. Louis, MO). GRASP55-green fluorescent protein (GFP) was made by generating a ClaI-XbaI insert of enhanced green fluorescent protein (EGFP) by polymerase chain reaction (PCR) and inserting it into ClaI-Xba1 sites of pCB6-GRASP55 (Jesch et al, 2001) to generate a C-terminal tag. Monolayer HeLa cells were grown and synchronized using a double thymidine block (48 h in thymidine and then 12 h without then 12 h in thymidine) as described previously (Feinstein and Linstedt, 2007).…”

“…GRASP55 is a likely target of this mitogen-activated protein kinase (MAPK) pathway because GRASP55 is a mitotic target of MEK-dependent phosphorylation by ERK at its T222 and T225 residues (Jesch et al, 2001) and because expression of an alanine-substituted version of GRASP55, GRASP55-T 222,225 A, blocks both G2 phase Golgi unlinking and mitotic entry (Feinstein and Linstedt, 2007). To directly test the role of GRASP55 in Golgi ribbon formation, we inhibited GRASP55 expression using RNAi, and we analyzed Golgi organization, membrane trafficking, and cell cycle progression.…”