Rho Signaling during Cytokinesis: The Labyrinth of Minotaur Among a plethora of known effectors of Rho proteins, four recently identified proteins were shown to be required for cytokinesis (Fig. 2). The formin-homology proteins, Drosophila Diaphanous (Castrillon and Wasserman 1994; Wasserman 1998) and its mouse homologue p140mDia1 (Watanabe et al. 1997), bind to and regulate profilin, an actin-binding protein that promotes F-actin polymerization and is required for cytokinesis (Giansanti et al. 1998; Suetsugu et al. 1999). Dictyostelium p21-activated serine/threonine kinase PAKa, a putative Cdc42/Rac effector, is thought to regulate myosin II assembly by inhibiting myosin II heavy chain kinase (Chung and Firtel 1999). Bovine Rho-associated kinase (cleavage furrow kinase) is required for the regulation of the contractile ring contractility and for phosphorylation of intermediate filaments, leading to their disassembly and segregation into daughter cells, which, in turn, ensure efficient cell separation (Kosako et al. 1997, Kosako et al. 1999; Yasui et al. 1998). Finally, mouse citron kinase functions at a later step by regulating actomyosin contraction in a Rho-dependent manner (Madaule et al. 1998). The role of these effectors in Rho signaling during cytokinesis is further suggested by their interaction with GTP-bound forms of Rho proteins, localization to the cleavage furrow, and colocalization with either Rho proteins or components of the contractile ring. However, a role of Rho-kinase and citron kinase in cytokinesis is suggested from experiments with dominant-negative mutants, and this conclusion awaits further proof in loss-of-function studies. How many Rho effectors does a cell need to undergo cytokinesis? Cytokinesis is a complex event involving assembly of actin, myosin, septins, and actin-interacting proteins into a contractile ring, its dynamic contraction, and disassembly at the end of cytokinesis. Most likely, these cytoskeletal events are regulated via several signaling pathways that converge on the contractile ring, with kinases featuring prominently among Rho effectors (Fig. 2). These pathways are likely to act cooperatively, as demonstrated recently for two Rho effectors, p140mDia1 and serine/threonine kinase ROCK, in the formation of actomyosin stress fibers (Watanabe et al. 1999). Rho-activated ROCK phosphorylates and inhibits myosin light chain (MLC) phosphatase, thus promoting accumulation of phosphorylated MLC generated by MLC kinase. Phosphorylated myosin II assembles into myosin filaments and associates with actin to form stress fibers. Interestingly, the kinase domain of citron shows the highest similarity to that of ROCK (Madaule et al. 1998), though it remains to be demonstrated if citron kinase regulates myosin II polymerization during cytokinesis. Since the contractile ring is a cortical structure more complex and dynamic than stress fibers, one can expect a high degree of complexity of Rho-mediated signaling pathways regulating its function. We propose that there is an elaborate hierarchy of proteins regulating cytoskeletal dynamics at the cleavage furrow, in particular polymerization/depolymerization of molecules making up the contractile ring. Since Rho proteins, their upstream regulators, and downstream effectors all localize at the cell equator during cytokinesis, the cleavage furrow is likely to function as a workshop where protein complexes that initiate and regulate cytokinesis are assembled and disassembled. The limited knowledge we have about signal transduction pathways that initiate and regulate cytokinesis tells us that there are at least two basic regulatory mechanisms operating during cytokinesis: (a) protein–protein interaction or binding of small molecules and (b) phosphorylation. Small G proteins undergo conformational change and become biologically active in response to GTP binding. A similar mechanism, involving protein–protein interaction, has been proposed recently for p140mDia1 (Watanabe et al. 1999). Binding of Rho•GTP to the Rho-binding domain of mDia1 is thought to disrupt the intramolecular interaction between protein termini releasing the FH1 and FH2 COOH-terminal domains required to induce actin polymerization. How common is such an activation mechanism? We know that Rho•GTP/effector interactions are necessary to initiate a signaling cascade. However, one can imagine that the signal may also be transduced via the formation of ternary protein complexes alone, without interaction-dependent conformational change as suggested for p140mDia1. Intermolecular interactions as well as conformational changes are likely to be featured in this “protein dance”. A second mechanism, likely to be universal, is regulation by phosphorylation. Kinases and phosphatases play prominent roles in downstream pathways (Rho effectors), but may also regulate the upstream components of the cytokinetic signaling machinery. ECT2 appears to be activated by phosphorylation which occurs specifically in G2/M phases and this phosphorylation is required for its exchange activity (Tatsumoto et al. 1999). Cdk1 or a Cdk1-regulated kinase may phosphorylate ECT2, since it contains several consensus phosphorylation sites for Cdk1 (Tatsumoto et al. 1999). Interestingly, other ECT2-related RhoGEFs implicated in cytokinesis also contain several Cdk1 phosphorylation sites, one of which is conserved in three species (amino acids [aa] 771–774 in Drosophila Pebble, aa 671–674 in mouse Ect2, and aa 814–817 in human ECT2). Finally, it is difficult to rationalize the unexpected cell cycle–dependent nuclear localization of three RhoGEFs required for cytokinesis in Drosophila and human cells (Prokopenko et al. 1999; Tatsumoto et al. 1999) or cell polarization in yeast (Cdc24p; Toenjes et al. 1999). All three proteins localize to the nucleus in interphase cells, their levels diminish before nuclear division (or upon nuclear envelope breakdown), and proteins reappear in divided nuclei. Is it evidence for a direct link between the cytokinetic machinery and the mitotic apparatus? Or do these proteins play some role in the nucleus that is unrelated to their roles in cytokinesis? Or is it just a common mechanism to inactivate a regulatory molecule by sequestering it into the nucleus (Pines 1999)? Answers to these and other questions await a better understanding of the molecular pathways initiating and regulating cytokinesis. |
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