Phosphorylation of two small GTP-binding proteins of the Rab family by p34cdc2

Abstract

Entry of a cell into mitosis induces a series of structural and functional changes including arrest of intracellular transport. Knowledge of how the mitotic cycle is driven progressed substantially with the identification of the p34cdc2 protein kinase as a subunit of maturation-promoting factor, the universal regulating component of the mitotic cycle. Activation of the kinase at the onset of mitosis is thought to trigger the important mitotic events by phosphorylating key proteins. Small guanine nucleotide-binding proteins have been implicated in regulating transport pathways. For instance, two small Ras-related GTP-binding proteins, Sec4p and Ypt1p, control distinct stages of the secretory pathway in budding yeast. The GTP-binding proteins of the Rab family in rats and humans display strong homologies with Sec4p and Ypt1p, and might therefore also be involved in regulating intracellular transport. Indeed, distinct Rab proteins are located in the exocytotic and endocytotic compartments. Interruption of vesicular transport during mitosis might involve modification of these proteins. We now present biochemical evidence for a mitosis-specific p34cdc2 phosphorylation of Rab1Ap and Rab4p. By contrast, Rab2p and Rab6p are not phosphorylated. We also show that the distribution of Rab1Ap and Rab4p between cytosolic and membrane-bound forms is different in interphase and mitotic cells. This may provide a clue to the mechanism by which phosphorylation could affect membrane traffic during mitosis.

A Rab1 mutant affecting guanine nucleotide exchange promotes disassembly of the Golgi apparatus.

“…Our results provide support for, but do not prove, that this may be the case. Intriguingly, rabla was previously shown to be phosphorylated by p34 ~,~ kinase, and its membrane association slightly enhanced in mitotic HeLa cells (3). However, we have been unable to obtain any further evidence relevant to this point from analysis of rabla mutants defective in phosphorylation (Davidson, H. W., and W. E. Balch, unpublished observations).…”

Cell cycle regulated expression and phosphorylation of hpttg proto-oncogene product

“…In vitro phosphorylation was performed as described (Bailly et al, 1991). Histone H1 (3 mg) or puri®ed 6His-hPTTG proteins (3 mg) were incubated in a 15 ml ®nal volume of TKM bu er (50 mM Tris-HCl, pH 7.4, 25 mM KCl, 5 mM MgCl 2 and 0.5 mM DTT) containing 100 mM cold ATP and 1.5 mCi [g-32 P]ATP (Amersham, 5000 Ci/mmol).…”

Golgi Ribbon Unlinking: An Organelle-Based G2/M Checkpoint

“…Golgi fragmentation has been reported under many experimental conditions that compromise ER export or ER-Golgi transport. 23,37,63 Therefore, the mitotic phosphorylation of proteins involved in membrane trafficking along the early secretory pathway, such as Rab1, 38 Golgin-84, 74 GM130 39-40 or p47 44 could slow down ER-Golgi transport (by blocking COPI vesicle budding and fusion) contributing to Golgi ribbon fragmentation. Last, it is also possible that microtubules and actin cytoskeleton, which are tightly associated to the Golgi architecture (reviewed in refs.…”

“…22,25 This effect is likely due to the cessation of ER-Golgi and intra-Golgi transport that is observed in metaphase leading partly to an accumulation of COPI vesicles. [35][36][37] Several CDK1-phosphorylated substrates functioning in the early secretory pathway, such as Rab1, 38 GM130, [39][40] GRASP65 [41][42][43] and p47, 44 could contribute to this.…”