Mitogen-activated Protein Kinase Kinase 1-dependent Golgi Unlinking Occurs in G₂Phase and Promotes the G₂/M Cell Cycle Transition

Abstract

Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G2/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle “checkpoint.” A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G2 to control G2/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G2 Golgi unlinking and the G2/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.

GRASP55 Regulates Golgi Ribbon Formation

“…The observed phenotype was similar to that previously noted after GM130 or GRASP65 knockdown (Puthenveedu et al, 2006), and quantification using the same image analysis protocol indicated a similar degree of Golgi fragmentation ( Figure 1C). The analysis was then carried out for cells arrested at late G2 phase of the cell cycle when the ribbon undergoes MEKdependent unlinking (Feinstein and Linstedt, 2007). As expected, control cells exhibited an unlinked Golgi, and the appearance of the Golgi in these G2 cells was indistinguishable from cells depleted of GRASP55 and fixed at either S phase or G2 phase (Figure 1, B and C).…”

“…Golgi ribbons become unlinked during late G2 phase Feinstein and Linstedt, 2007) and a block in Golgi disassembly arrests or delays the G2/M transition (Sutterlin et al, 2002;Feinstein and Linstedt, 2007). Premitotic Golgi unlinking depends in part on the constitutive membrane fission activity of CtBP/ BARS (Hidalgo Carcedo et al, 2004; Abbreviations used: CDK, cyclin-dependent protein kinase; GalNAcT2, N-acetylgalactosaminyl transferase-2; GFP, green fluorescent protein; GM130, Golgi matrix protein 130 kDa; GRASP, Golgi reassembly protein; GST, glutathione transferase; MAPK, mitogen-activated protein kinase; RNAi, RNA interference; siRNA, small interfering RNA. …”

“…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).If the MEK inhibitor U0126 delays cyclin-dependent protein kinase (CDK)1 activation by preventing MEK/ERK inhibition of GRASP55, then GRASP55 knockdown would be expected to suppress this inhibition. As a test, control and GRASP55-depleted cells were synchronized, and then they were allowed to progress into M phase in the presence or absence of U0126.…”

“…© 2008 by The American Society for Cell Biology 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).…”

“…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.…”