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 Golgi-localized 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.

Figure 1

Comparison of human and rat GRASP55 sequences. Alignment of the human GRASP55 and rat GRASP55 (AF110267) sequences. Capital letters indicate exact identity. The underlined region designates the amino-terminus that was cloned for this study. Potential sites of proline-directed phosphorylation are boxed. The ideal ERK consensus motif is shown in bold, and residues that were mutated to alanine are indicated with asterisks.

Figure 2

Human GRASP55 is a Golgi-localized MPM2 antigen. (A) HeLa cells were transfected with human GRASP55-MYC and processed for double-label immunofluorescence microscopy. The GRASP55-MYC localization pattern as indicated by anti-myc staining is shown. (B) The staining pattern of the Golgi protein giantin in the same cell. Bar, 50 μm. (C) Unsynchronized (I) or mitotic (M) HeLa cells that were mock-transfected (lanes 1 and 2), wild-type (wt) GRASP55-MYC transfected (lanes 3 and 4), or T222A T225A GRASP55-MYC transfected (lanes 5 and 6) were subjected to immunoprecipitation with the anti-myc antibody. The resulting precipitates were analyzed for MPM2 reactivity by immunoblotting. (D) The immunoblot was reprobed with anti-myc antibody to determine GRASP55-MYC recovery. The transfected protein was recovered from both mitotic and interphase cells but only mitotic GRASP55-MYC was MPM2 reactive. As determined by densitometry and after normalization to account for the small difference in protein recovery, the MPM2 reactivity of T222A T225A GRASP55-MYC was 9% that of GRASP55-MYC.

Figure 3

In vitro mitotic phosphorylation of GRASP55 on consensus ERK sites. (A) MPM2 reactivity of GST-GRASP55 was determined after an in vitro incubation with either interphase (I) or mitotic (M) HeLa cell extracts in the presence or absence of added ATP. One sample (lane 4) was treated with potato acid phosphatase before MPM2 immunoblotting. MPM2 reactivity required ATP and mitotic extract and was lost with phosphatase treatment. (B) After in vitro incubation with interphase or mitotic extracts in the presence of added ATP, the MPM2 reactivity was determined for wild-type (wt) GST-GRASP55 and a version containing T222A and T225A substitutions. Mutation of consensus ERK sites in GRASP55 blocked MPM2 reactivity.

Figure 4

Inhibition of ERK activity by U0126, a specific inhibitor of MKK, inhibits GRASP55 phosphorylation. (A) MPM2 reactivity of GST-GRASP55 was determined after in vitro incubation with added ATP and mitotic cell extracts that had been pretreated with either 0.2% DMSO control or 20 μM U0126 for 30 min at 37°C. (B) ³²P incorporation of transfected GRASP55-MYC recovered by immunoprecipitation from mitotic HeLa cells that were pretreated for 30 min at 37°C with either 0.2% DMSO control or 20 μM U0126 and then incubated for 4 h with [³²P]H₃PO₄.

Figure 5

GRASP55 is directly phosphorylated by active MAPKs. MPM2 reactivity of equivalent amounts of wild-type (wt) or the alanine-substituted (T222A T225A) GST-GRASP55 was determined after in vitro incubation, 30 min at 37°C, with an ATP containing buffer and purified active ERK2, JNK, p38, or MKK1 (G7B, ΔN4/S218D/S222D). An equivalent amount of each active kinase was used in each assay.