Evidence for a Role of CLIP-170 in the Establishment of Metaphase Chromosome Alignment

CLIPs (cytoplasmic linker proteins) are a class of proteins believed to mediate the initial, static interaction of organelles with microtubules. CLIP-170, the CLIP best characterized to date, is required for in vitro binding of endocytic transport vesicles to microtubules. We report here that CLIP-170 transiently associates with prometaphase chromosome kinetochores and codistributes with dynein and dynactin at kinetochores, but not polar regions, during mitosis. Like dynein and dynactin, a fraction of the total CLIP-170 pool can be detected on kinetochores of unattached chromosomes but not on those that have become aligned at the metaphase plate. The COOH-terminal domain of CLIP-170, when transiently overexpressed, localizes to kinetochores and causes endogenous full-length CLIP-170 to be lost from the kinetochores, resulting in a delay in prometaphase. Overexpression of the dynactin subunit, dynamitin, strongly reduces the amount of CLIP-170 at kinetochores suggesting that CLIP-170 targeting may involve the dynein/dynactin complex. Thus, CLIP-170 may be a linker for cargo in mitosis as well as interphase. However, dynein and dynactin staining at kinetochores are unaffected by this treatment and further overexpression studies indicate that neither CLIP-170 nor dynein and dynactin are required for the formation of kinetochore fibers. Nevertheless, these results strongly suggest that CLIP-170 contributes in some way to kinetochore function in vivo.Microtubules (MTs)¹ in vertebrate somatic cells are involved in intracellular transport and distribution of membranous organelles. Fundamental to this role are their tightly controlled, polarized organization, and unusual dynamic properties (Hirokawa, 1994) and their interaction with a complex set of MT-based motor proteins (Hirokawa, 1996; Sheetz, 1996; Goodson et al., 1997). During mitosis, they contribute to the motility of centrosomes, the construction of spindle poles (Karsenti et al., 1996; Merdes and Cleveland, 1997), and the dynamic movements of kinetochores (Rieder and Salmon, 1994) and chromosome arms (Barton and Goldstein, 1996; Vernos and Karsenti, 1996). The motor protein cytoplasmic dynein, drives the transport toward MT minus-ends of a variety of subcellular organelles (Schnapp and Reese, 1989; Schroer et al., 1989; Holzbaur and Vallee, 1994). Dynactin is a molecular complex originally identified as being essential for dynein-mediated movement of salt-washed vesicles in vitro (reviewed in Schroer, 1996; Schroer and Sheetz, 1991). Genetic studies in fungi, yeast, and flies have shown that the two complexes function together to drive nuclear migration, spindle and nuclear positioning and to permit proper neuronal development (Eshel et al., 1993; Clark and Meyer, 1994; Muhua et al., 1994; Plamann et al., 1994; McGrail et al., 1995; Karsenti et al., 1996). Biochemical studies suggest a direct interaction between certain subunits of dynein and dynactin (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995). In vivo, the two molecules may bind one another transiently, since they have not been isolated as a stable complex.There is good evidence indicating that the dynein/dynactin complex, together with other motors (Eg5, and a minus-end oriented kinesin-related protein) and a structural protein (NuMa), drive the focusing of free microtubule ends into mitotic spindle poles (Merdes and Cleveland, 1997; Waters and Salmon, 1997)_. A trimolecular complex composed of NuMa and dynein/dynactin may be crucial in this process in both acentriolar (Merdes et al., 1996), and centriolar spindles (Gaglio et al., 1997). A number of findings also indicate that the combined actions of dynein and dynactin at the kinetochore contribute to chromosome alignment in vertebrate somatic cells. First, the initial interaction between polar spindle MTs and kinetochores seems to involve a tangential capture event (Merdes and De Mey, 1990; Rieder and Alexander, 1990) which is followed by a poleward gliding along the surface lattice of the MT (Hayden et al., 1990). Both in vivo and in vitro (Hyman and Mitchison, 1991) this gliding movement appears similar to the dynein-mediated retrograde transport of vesicular organelles along MTs. Consistent with this is the finding that both dynein (Pfarr et al., 1990; Steuer et al., 1990) and its activator, dynactin (Echeverri et al., 1996), are present at prometaphase kinetochores. Overexpression of dynamitin, a 50-kD subunit of the dynactin complex, results in the partial disruption of the dynactin complex and in the loss, from kinetochores, of dynein, as well as dynactin. Therefore, it has been proposed that dynactin mediates the association of dynein with kinetochores. Abnormal spindles with poorly focused poles are observed and the cells become arrested in pseudoprometaphase (Echeverri et al., 1996). Despite these findings, rigorous proof for a role of the dynein motor complex in kinetochore motility is still lacking, and its role may differ between lower and higher eucaryotes, and between mitosis and meiosis.CLIP-170 (Rickard and Kreis, 1996) is needed for in vitro binding of endocytic transport vesicles to MTs (Pierre et al., 1992). It is a nonmotor MT-binding protein that accumulates preferentially in the vicinity of MT plus ends and on early endosomes and endocytic transport vesicles in nondividing cells (Rickard and Kreis, 1990; Pierre et al., 1992). Like many MT-binding proteins, CLIP-170 is a homodimer whose NH₂-terminal head domains and COOH-terminal tail domains flank a central α-helical coiled-coil domain. The binding of CLIP-170 to MTs involves a 57–amino acid sequence present twice in the head domain (Pierre et al., 1992) and is regulated by phosphorylation (Rickard and Kreis, 1991). The COOH-terminal domain has been proposed to participate in targeting to endocytic membranes (Pierre et al., 1994). The fact that the latter move predominantly toward microtubule minus ends in a process most likely mediated by cytoplasmic dynein and dynactin (Aniento and Gruenberg, 1995), suggests that CLIP-170 may act in concert with this motor complex, and may be subject to regulated interactions with one or more dynactin or dynein subunits at the vesicle membrane.Here we report that during mitosis, CLIP-170 codistributes with dynein and dynactin at kinetochores, but not spindle poles. Evidence is presented that the COOH-terminal domain of CLIP-170 is responsible for its kinetochore targeting, and that this may be mediated by the complex of dynein and dynactin. The effects on mitotic progression of overexpression of wild type and several deletion mutants of CLIP-170 provide evidence for the involvement of CLIP-170 in kinetochore function early in mitosis. We also present in vivo evidence that neither CLIP-170 nor the complex of dynein and dynactin are required for formation of kinetochore fibers.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Systems-wide Analysis of K-Ras,Cdc42, and PAK4 Signaling by Quantitative Phosphoproteomics

Although K-Ras, Cdc42, and PAK4 signaling are commonly deregulated in cancer, only a few studies have sought to comprehensively examine the spectrum of phosphorylation-mediated signaling downstream of each of these key signaling nodes. In this study, we completed a label-free quantitative analysis of oncogenic K-Ras, activated Cdc42, and PAK4-mediated phosphorylation signaling, and report relative quantitation of 2152 phosphorylated peptides on 1062 proteins. We define the overlap in phosphopeptides regulated by K-Ras, Cdc42, and PAK4, and find that perturbation of these signaling components affects phosphoproteins associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. These findings provide a resource for future studies to characterize novel targets of oncogenic K-Ras signaling and validate biomarkers of PAK4 inhibition.The Ras oncoproteins are small monomeric GTPases that transduce mitogenic signals from cell surface receptor tyrosine kinases (RTKs) to intracellular serine/threonine kinases. Approximately thirty percent of human tumors harbor a somatic gain-of-function mutation in one of three RAS genes, resulting in the constitutive activation of Ras signaling and the aberrant hyperactivation of growth-promoting effector pathways (1). Designing therapeutic agents that directly target Ras has been challenging (2, 3), and thus clinical development efforts have focused on targeting effector pathways downstream of Ras. The Raf-MEK-ERK and PI3K-Akt effector pathways have been extensively studied and several small molecule inhibitors targeting these pathways are currently under clinical evaluation (4, 5). However, biochemical studies and mouse models indicate that several additional effector pathways are essential for Ras-driven transformation and tumorigenesis (6–11). Hence, a comprehensive characterization of these effector pathways may reveal additional druggable targets.The Rho GTPase Cdc42 lies downstream of Ras (12–14) and regulates many cellular processes that are commonly perturbed in cancer, including migration, polarization, and proliferation (15) (Fig. 1A). Importantly, Cdc42 is overexpressed in several types of human cancer (16–20) and is required for Ras-driven cellular transformation (13, 21, 22). Recent studies show that genetic ablation of Cdc42 impairs Ras-driven tumorigenesis (13), indicating the potential of Cdc42 and its effectors as drug targets in Ras mutant tumors.Open in a separate windowFig. 1.Experimental workflow. A, K-Ras is a small GTPase that regulates the activity of a variety of downstream proteins including the Rho GTPase Cdc42. The PAK4 serine/threonine kinase is a direct effector of Cdc42 and regulates actin reorganization, microtubule stability, and cell polarity. B, To measure large-scale phosphorylation changes induced by constitutive K-Ras or Cdc42 signaling or PAK4 ablation, the quantitative label-free PTMscan® approach was employed (Cell Signaling Technology). Briefly, for each condition extracted proteins were digested with trypsin and separated from non-peptide material by solid-phase extraction with Sep-Pak C18 cartridges. Three phosphorylation motif antibodies were used serially to isolate phosphorylated peptides in independent immunoaffinity purifications (CDK substrate motif [K R]-pS-P-X-[K R], CK substrate motif pT-[D E]-X-[D E], PKD substrate motif l-X-R-X-X-p[S T]). The samples were run in duplicate and tandem mass spectra were collected with an LTQ-Orbitrap hybrid mass spectrometer. pLPC is an empty vector control.In particular, the p21-activated kinases (PAKs) are Cdc42 effectors that have generated significant interest (23, 24), as they are central components of key oncogenic signaling pathways and regulate cytoskeletal organization, cell migration, and nuclear signaling (25). The PAK family is comprised of six members and is subdivided into two groups (Groups I and II) based on sequence and structural homology. Group I PAKs (PAK1–3) are relatively well characterized, however, much less is known regarding the function and regulation of Group II PAKs (PAK4–6). The kinase domains of Group I and II PAKs share only about 50% identity, suggesting the two groups may recognize distinct substrates and govern unique cellular processes (26).The Group II PAK family member PAK4 is of particular interest as it is overexpressed or genetically amplified in several lung, colon, prostate, pancreas, and breast tumor cell lines and samples (26–30). Furthermore, functional studies have implicated PAK4 in cell transformation, cell invasion, and migration (27, 31). Xenograft studies in athymic mice show an important role for PAK4 in mediating Cdc42- or K-Ras-driven tumor formation, highlighting a critical role for Pak4 downstream of these GTPases (32). Given its roles in transformation, tumorigenesis, and oncogenic signaling, there is significant interest in targeting PAK4 therapeutically (23). PAK4 binds and phosphorylates several proteins involved in cytoskeletal organization and apoptosis, including Lim domain kinase 1 (LIMK1) (33), guanine nucleotide exchange factor-H1 (GEF-H1) (34), Raf-1 (35), and Bad (36). However, the Group I PAK family member PAK1 also phosphorylates several of these PAK4 targets (37). Thus, there remains a need to identify robust and selective pharmacodynamic biomarkers for PAK4 inhibition.Despite the importance of PAK4 and its upstream regulators in cancer development, few studies have sought to comprehensively characterize the spectrum of K-Ras, Cdc42, or PAK4 mediated phosphorylation signaling (37–39). Recent developments in mass spectrometry allow the in-depth identification and quantitation of thousands of phosphorylation sites (40–43). The majority of large-scale efforts have aimed to identify the basal phosphoproteomes of different species (44, 45) or tissues (46) to characterize global steady-state phosphorylation. However, this methodology can also be applied to quantify perturbed phosphorylation regulation in cancer signaling pathways (40, 47–49), and has the potential to reveal novel biomarkers of oncogenic signaling.In this study, we completed a label-free quantitative analysis of K-Ras, Cdc42, and PAK4 phosphorylation signaling using the PTMScan® method, which has proven as robust and reproducible quantitation technology (50, 51). We quantified phosphorylation levels in wild-type and PAK4 knockout NIH3T3 cells expressing oncogenic K-Ras, activated Cdc42, or an empty vector control to elucidate the molecular pathways and functions modulated by these key signaling proteins. We report relative quantitation of 2152 phosphorylated peptides on 1062 proteins among the different conditions, and find that many of the regulated phosphoproteins are associated with microtubule depolymerization, cytoskeletal organization, and the cell cycle. To our knowledge, our study is the first to examine the overlap among signaling networks regulated by K-Ras, Cdc42, and PAK4, and provides a resource for future studies to further interrogate the perturbation of this signaling pathway.     

A +TIP for a smooth trip

Is there a cellular mechanism for preventing a depolymerizing microtubule track from “slipping out from under” its cargo? A recent study in budding yeast indicates that when a chromosome is transported to the minus end of a spindle microtubule, its kinetochore-bound microtubule plus end–tracking protein (+TIP) Stu2 may move to the plus end to promote rescue; i.e., to switch the depolymerizing end to a polymerizing end. The possibility that other +TIPs may play a similar role in sustaining a microtubule track during vesicular transport deserves investigation.Microtubule motor proteins such as dynein and kinesins are responsible for transporting cellular cargos along microtubule tracks (Vale, 2003). The net direction and speed of cargo movement, however, are likely to be regulated in a very complicated fashion, especially when a cargo is bound to multiple motors with opposite directionalities (Vale 2003; Mallik and Gross 2004; Levi et al., 2006). The fact that the microtubule track is not very stable further complicates matters. The plus ends of microtubules, which face the cell periphery in most cell types, are highly dynamic, exhibiting alternating periods of polymerization (growth) and depolymerization (shrinkage; Desai and Mitchison, 1997). Such plus end dynamics may be useful for searching and capturing relatively stationary cargos near the cell periphery that need to be transported inward (Vaughan et al., 2002). However, the dynamic nature of the track can also create an obvious problem for the transport process. If a microtubule''s rate of shrinkage is greater than the rate of cargo transport, then the microtubule may shrink past an attached cargo, causing its dissociation from the track. Does this happen in cells, or do cells have a mechanism to prevent it?Although this question has never been directly addressed, a recent study on budding yeast chromosome segregation has shed new light on the issue (Tanaka et al., 2005). In this study, the authors took advantage of a strategy that allowed them to specifically shut off the function of a single kinetochore, thereby preventing it from attaching to a spindle microtubule while, at the same time, permitting other kinetochores to attach to the spindle. After the function of this single kinetochore was switched back on, the behavior of its associated chromosome on a spindle microtubule was subjected to a detailed image analysis. Several important insights from this study on chromosome–microtubule interactions during mitosis have been recently reviewed (Bloom 2005), and, thus, only those observations that pertain to cargo transport will be highlighted here. The chromosome was first seen to undergo a lateral interaction with the microtubule followed by minus end–directed transport toward the pole. The mechanism of the minus end–directed transport is not entirely clear, although a member of the kinesin-14 family, Kar3, may be one of the players in this process (Tanaka et al., 2005). During transport, the attached microtubule can undergo shrinkage with a rate higher than that of the minus end–directed chromosome movement (Tanaka et al., 2005); however, it never shrank beyond the position of the cargo. Such exquisite control over the extent of shrinkage appears to rely on a conversation between the cargo and the plus end of the microtubule that is mediated by the microtubule plus end–tracking protein Stu2 (Bloom 2005; Tanaka et al., 2005).Microtubule plus end–tracking proteins (+TIPs) are a class of proteins that use different structural motifs or specific targeting mechanisms to localize to the dynamic plus ends of microtubules (Carvalho et al., 2003; Akhmanova and Hoogenraad, 2005). Although most +TIPs associate with only the growing ends of microtubules, several +TIPs also localize to the shrinking ends (Carvalho et al., 2003; 2004; Akhmanova and Hoogenraad, 2005; Mennella et al., 2005; Sproul et al., 2005; Molk et al., 2006; Wu et al., 2006). Many +TIPs have been found to impact microtubules by either promoting their growth or promoting dynamic behavior. Stu2 is a member of the XMAP215/TOG/Dis1/DdCP224 family of proteins that have been shown to affect microtubule dynamics in multiple ways depending on different experimental conditions (Ohkura et al., 2001; Popov and Karsenti 2003; Holmfeldt et al., 2004; Akhmanova and Hoogenraad, 2005). In vitro, Stu2 binds to the plus ends of preformed microtubules and promotes catastrophe, which is a switch from growth to shrinkage (van Breugel et al., 2003). In vivo studies using mutants of Stu2, however, indicate that Stu2 promotes microtubule growth (Severin et al., 2001) and the dynamics of both kinetochore and cytoplasmic microtubules (Kosco et al., 2001; Pearson et al., 2003). During anaphase B spindle elongation, Stu2 may antagonize the function of Kip3 (a kinesin-13 family member) to promote the plus end polymerization of overlapping microtubules (Severin et al., 2001). Although it is not fully understood how or why Stu2 is so versatile, it is well recognized that the in vivo interactions among +TIPs are very complicated, and the loss of function of a +TIP in vivo may decrease or increase the accumulation of other +TIPs that also regulate microtubule dynamics (Carvalho et al., 2003; 2004; Lansbergen et al., 2004; Akhmanova and Hoogenraad, 2005; Galjart 2005; Komarova et al., 2005). Tanaka et al. (2005) identified Stu2 as a rescue (a switch from shrinkage to growth) factor based not on phenotypic studies of Stu2 mutants but, instead, on a direct observation of the relationship between microtubule plus end behavior and Stu2 localization. They found that Stu2 was localized at the plus ends of microtubules emanating from the spindle pole body, and, during periods of microtubule shrinkage, Stu2 levels at the plus ends were decreased. Interestingly, Stu2 was also localized at the unbound kinetochore. When the kinetochore subsequently attached laterally to a spindle microtubule and underwent minus end–directed transport, the Stu2 proteins were transported from the kinetochore to the microtubule plus end. The arrival of Stu2 at the plus end closely correlated to the rescue of the shrinking microtubule (Tanaka et al., 2005). These observations strongly suggest that the Stu2 carried by the kinetochore may serve as a rescue factor for the microtubule track, preventing it from vanishing before the migrating chromosome.Could such a scenario exist during microtubule-dependent transport of nonchromosomal cargoes during interphase? We do not yet know the answer. However, based on published studies, it seems reasonable to hypothesize that other +TIPs, especially the cytoplasmic linker protein CLIP-170, may function in a manner similar to yeast Stu2 to ensure a safe trip for a minus end–directed cargo. CLIP-170 contains CAP-Gly microtubule-binding motifs at its NH₂ terminus and was initially identified as a protein required for linking endocytic vesicles to microtubules in vitro (Pierre et al., 1992; Rickard and Kreis 1996). Later, CLIP-170 was identified as a founding member of the microtubule plus end–tracking proteins (Perez et al., 1999). The connection between CLIP-170''s in vitro endosome–microtubule linking property and its in vivo plus end tracking behavior has not been clearly made. Could an endocytic vesicle use its bound CLIP-170 as a rescue factor to prevent the disappearance of the track on which it is traveling?CLIP-170 is indeed considered to be a rescue factor in mammalian cells (Komarova et al., 2002a). Komarova et al. (2002b) have found that in cultured CHO and NRK cells, microtubule dynamics seem to be controlled spatially; catastrophe and rescue occur frequently only near the cell periphery. Although the mechanisms behind catastrophe and rescue are not fully understood, protein factors are required for regulating both events in vivo (Desai and Mitchison, 1997). In CHO cells, a dominant-negative form of CLIP-170 that displaces the endogenous CLIP-170 from microtubule plus ends severely reduces the rescue frequency so that microtubules are more likely to shrink all the way back to the microtubule-organizing center (Komarova et al., 2002a). Moreover, both in vivo and in vitro studies suggest that the rescue activity of CLIP-170 is localized to the NH₂ terminus containing the CAP-Gly motifs (Komarova et al., 2002a; Arnal et al., 2004). How CLIP-170 rescues a shrinking end is not clear. CLIP-170 can promote tubulin oligomerization (Diamantopoulos et al., 1999; Arnal et al., 2004), and it is likely that this property serves to increase the local concentration of tubulin substrate, thereby lowering the entropic barrier for the polymerization reaction. CLIP-170 in mammalian cells has only been found at growing plus ends, most likely as a result of copolymerization with tubulin subunits followed by its release from older segments (Diamantopoulos et al., 1999; Perez et al., 1999; Folker et al., 2005). When a microtubule end shrinks, CLIP-170 falls off. Is there a mechanism to get CLIP-170 close to the depolymerizing end and facilitate its function as a rescue factor? Given the proposed function of Stu2 as a rescue factor for spindle microtubules, one may easily imagine a similar scenario in which vesicle-bound CLIP-170 may be transported to the approaching microtubule end to rescue it from further shrinkage.If vesicle-bound CLIP-170 is transported to the plus end in a manner similar to Stu2, could such transport be mediated by plus end–directed kinesins? Although the kinesin involved in transporting Stu2 toward the microtubule plus end still needs to be identified, detailed image analyses have revealed a role for the Kip2/Tea2 kinesins (members of the kinesin-7 family) in transporting CLIP-170 homologues in fungi (Busch et al., 2004; Carvalho et al., 2004). Bik1 and Tip1 are the CLIP-170 homologues in budding and fission yeasts, respectively, and these proteins are found at microtubule plus ends, where they act as growth-promoting factors or anticatastrophe factors (Berlin et al., 1990; Brunner and Nurse 2000; Carvalho et al., 2004). In both yeasts, the Kip2/Tea2 kinesins bind to and comigrate with the CLIP-170 homologues along the microtubule toward the plus end (Busch et al., 2004; Carvalho et al., 2004). Kinesins have also been implicated in targeting other +TIPs to microtubule plus ends (Jimbo et al., 2002; Maekawa et al., 2003; Zhang et al., 2003; Wu et al., 2006). For example, the mammalian tumor suppressor protein APC (adenomatous polyposis coli) may be targeted to the plus end by KIF3A/KIF3B (a heterotrimeric kinesin II in the kinesin-2 family) as well as by other mechanisms (Jimbo et al., 2002; Nathke 2004; Slep et al., 2005). It will be interesting to see whether a similar transport process for CLIP-170 exists in higher eukaryotic cells. It is possible that such a mechanism would deliver just enough CLIP-170 to the shrinking plus end to initiate rescue. When microtubule growth is resumed, CLIP-170''s intrinsic higher affinity for tubulin subunits and lower affinity for the microtubule wall may allow these proteins to “treadmill” on the growing end (Perez et al., 1999; Folker et al., 2005).The regulation of CLIP-170 activity appears to be rather complex. CLIP-170 is most likely phosphorylated by multiple kinases, including FKBP12–rapamycin-associated protein (mTOR; Choi et al., 2002). Although phosphorylation by mTOR/FKBP 12–rapamycin-associated protein may stimulate CLIP-170''s microtubule binding, phosphorylation by other kinases may cause CLIP-170 to dissociate from microtubules (Rickard and Kreis 1996; Choi et al., 2002). In vivo, CLIP-170 has a closed conformation that is presumably inactive and an open conformation that may interact with microtubules and dynein regulators such as dynactin (Schroer 2004) and LIS1 (Morris et al., 1998; Lansbergen et al., 2004). It is possible that phosphorylation may regulate the conversion between these two forms, but the specific mechanism and the spatial regulation for this conversion have yet to be resolved. If CLIP-170 is indeed released from a membranous cargo to move to the plus end in order to serve as a rescue factor, it would be interesting to know when and/or where such a conformational switch occurs. Finally, other proteins may play redundant roles with CLIP-170 in vesicular trafficking, which may explain why a dramatic defect in vesicle/organelle distribution is not detected when the CLIP-170 level is lowered or when the gene is knocked out (Lansbergen et al., 2004; Akhmanova et al., 2005).+TIPs other than CLIP-170 may play a similar role in rescuing shrinking microtubule tracks. For example, the dynactin complex that links dynein to membranous cargoes and promotes the processive motion of dynein (Schroer 2004) may act as a rescue factor. The p150^Glued subunit of dynactin and CLIP-170 both contain CAP-Gly microtubule-binding motifs at their NH2 termini, although p150^Glued contains one, whereas CLIP-170 contains two such motifs. Dynactin has been shown to behave as a +TIP facilitating the capture of vesicular cargo for minus end–directed transport (Vaughan et al., 1999; 2002). The head domain of the p150^Glued subunit containing the CAP-Gly motif has been shown to promote rescue in vivo in the absence of endogenous CLIP-170, although the effect was much weaker than that caused by the exogenous CLIP-170 head domain (Kamarova et al., 2002a). In vitro studies showed that dynactin may promote nucleation during microtubule assembly (Ligon et al., 2003), which is consistent with it being a potential rescue factor. As shown with CLIP-170, this capacity to bring multiple tubulins together may help to overcome the entropic barrier of the polymerization reaction. Finally, cargo-bound dynactin may also use kinesin to get to the plus end. The p150^Glued subunit of dynactin has been shown to interact directly with the COOH terminus of KAP3, a subunit of the heterotrimeric kinesin II (a member of the kinesin-2 family) that also binds to APC (Jimbo et al., 2002; Deacon et al., 2003; Dell 2003). Although this binding is implicated in dynactin''s role as a cargo adaptor for kinesin II, it is possible, in theory, that a small amount of dynactin may use this connection to move to the plus end.The proposed hypothesis that +TIPs may be released from a membranous cargo to rescue a shrinking microtubule track may apply to both minus and plus end–directed transport. In addition, it is important to point out that this hypothesis does not exclude other mechanisms for rescuing long microtubule tracks. Rescue may occur stochastically, and, sometimes, +TIPs may participate in other ways such as mediating microtubule capture by the actin-rich cortex to stabilize the track (Wen et al., 2004; Galjart 2005). In some situations, microtubule dynamics are modulated by the direct binding of membranous cargo to the growing or shrinking plus ends of microtubules (Waterman-Storer and Salmon, 1998).Currently, the ability of CLIP-170 or other +TIPs to be released from a membranous cargo and to act as a rescue factor for a shrinking microtubule is just a hypothesis. Nevertheless, searching for proteins involved in the communication between a cargo and the approaching shrinking end of its microtubule track is clearly an endeavor worth pursuing.     

Regulation of Microtubule Dynamic Instability in Vitro by Differentially Phosphorylated Stathmin

Stathmin is an important regulator of microtubule polymerization and dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule dynamic instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser¹⁶ or Ser⁶³, and doubly phosphorylated at Ser²⁵ and Ser³⁸, on its ability to modulate microtubule dynamic instability at steady-state in vitro. Phosphorylation at either Ser¹⁶ or Ser⁶³ strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on dynamic instability are strongly but differently attenuated by phosphorylation at Ser¹⁶ and Ser⁶³ and support the hypothesis that selective targeting by Ser¹⁶-specific or Ser⁶³-specific kinases provides complimentary mechanisms for regulating microtubule function.Stathmin is an 18-kDa ubiquitously expressed microtubule-destabilizing phosphoprotein whose activity is modulated by phosphorylation of its four serine residues, Ser¹⁶, Ser²⁵, Ser³⁸, and Ser⁶³ (1–7). Several classes of kinases have been identified that phosphorylate stathmin, including kinases associated with cell growth and differentiation such as members of the mitogen-activated protein kinase (MAPK)2 family, cAMP-dependent protein kinase (1–5, 8–11), and kinases associated with cell cycle regulation such as cyclin-dependent kinase 1 (3, 12–14). Phosphorylation of stathmin is required for cell cycle progression through mitosis and for proper assembly/function of the mitotic spindle (3, 13–16). Inhibition of stathmin phosphorylation produces strong mitotic phenotypes characterized by disassembly and disorganization of mitotic spindles and abnormal chromosome distributions (3, 13–14).Stathmin is known to destabilize microtubules in two ways. One is by binding to soluble tubulin and forming a stable complex that cannot polymerize into microtubules, consisting of one molecule of stathmin and two molecules of tubulin (T2S complex) (17–24). Addition of stathmin to microtubules in equilibrium with soluble tubulin results in sequestration of the tubulin and a reduction in the level of microtubule polymer (17–18, 22, 25–28). In addition to reducing the amount of assembled polymer, tubulin sequestration by stathmin has been shown to increase the switching frequency at microtubule plus ends from growth to shortening (called the catastrophe frequency) as the microtubules relax to a new steady state (17, 29). The second way is by binding directly to microtubules (27–30). The direct binding of stathmin to microtubules increases the catastrophe frequency at both ends of the microtubules and considerably more strongly at minus ends than at plus ends (27). Consistent with its strong catastrophe-promoting activity at minus ends, stathmin increases the treadmilling rate of steady-state microtubules in vitro (27). These results have led to the suggestion that stathmin might be an important cellular regulator of minus-end microtubule dynamics (27).Phosphorylation of stathmin diminishes its ability to regulate microtubule polymerization (3, 14, 25–26). Phosphorylation of Ser¹⁶ or Ser⁶³ appears to be more critical than phosphorylation of Ser²⁵ and Ser³⁸ for the ability of stathmin to bind to soluble tubulin and to inhibit microtubule assembly in vitro (3, 25). Inhibition of stathmin phosphorylation induces defects in spindle assembly and organization (3, 14) suggesting that not only soluble tubulin-microtubule levels are regulated by phosphorylation of stathmin, but the dynamics of microtubules could also be regulated in a phosphorylation-dependent manner.It is not known how phosphorylation at any of the four serine residues of stathmin affects its ability to regulate microtubule dynamics and, specifically, its ability to increase the catastrophe frequency at plus and minus ends due to its direct interaction with microtubules. Thus, we determined the effects of stathmin individually phosphorylated at either Ser¹⁶ or Ser⁶³ and doubly phosphorylated at both Ser²⁵ and Ser³⁸ on dynamic instability at plus and minus ends in vitro at microtubule polymer steady state and physiological pH (pH 7.2). We find that phosphorylation of Ser¹⁶ strongly reduces the direct catastrophe-promoting activity of stathmin at plus ends and abolishes it at minus ends, whereas phosphorylation of Ser⁶³ abolishes the activity at both ends. The effects of phosphorylation of individual serines correlated well with stathmin''s reduced abilities to form stable T2S complexes, to inhibit microtubule polymerization, and to bind to microtubules. In contrast, double phosphorylation of Ser²⁵ and Ser³⁸ did not alter the ability of stathmin to modulate dynamic instability at the microtubule ends, its ability to form a stable T2S complex, or its ability to bind to microtubules. The data further support the hypotheses that phosphorylation of stathmin on either Ser¹⁶ or Ser⁶³ plays a critical role in regulating microtubule polymerization and dynamics in cells.     

Global Analysis of Cdc14 Dephosphorylation Sites Reveals Essential Regulatory Role in Mitosis and Cytokinesis

Degradation of the M phase cyclins triggers the exit from M phase. Cdc14 is the major phosphatase required for the exit from the M phase. One of the functions of Cdc14 is to dephosphorylate and activate the Cdh1/APC/C complex, resulting in the degradation of the M phase cyclins. However, other crucial targets of Cdc14 for mitosis and cytokinesis remain to be elucidated. Here we systematically analyzed the positions of dephosphorylation sites for Cdc14 in the budding yeast Saccharomyces cerevisiae. Quantitative mass spectrometry identified a total of 835 dephosphorylation sites on 455 potential Cdc14 substrates in vivo. We validated two events, and through functional studies we discovered that Cdc14-mediated dephosphorylation of Smc4 and Bud3 is essential for proper mitosis and cytokinesis, respectively. These results provide insight into the Cdc14-mediated pathways for exiting the M phase.All cells proliferate following a fixed, highly coordinated cycle. Mitosis especially requires elaborate coordination for proper chromosome segregation, mitotic spindle disassembly, and cytokinesis. Much of this activity is facilitated by numerous, diverse phosphorylation and dephosphorylation signals that orchestrate the precise progression of M phase.Prior to mitosis, sister chromatids resulting from DNA replication during S phase are held together by the cohesion complex. Then, during prophase, chromosomes are condensed by the condensin (Smc2/4) complex (1) and microtubules are remodeled to form the mitotic spindle (2). Subsequently, in metaphase, the microtubules of the spindle apparatus attach to the chromosome kinetochores (3) and dissolution of the sister chromatids is triggered by the separase-mediated cleavage of cohesin (4, 5). Finally, Cdc14, Cdh1, and APC/C work together in telophase to degrade the M phase cyclins (6), promote decondensation of chromosomes (7), and finish cytokinesis (8, 9).Cdc14, a dual-specificity phosphatase that removes the phosphate group on both phosphotyrosine and phosphoserine/threonine residues (10), is required for mitosis (11, 12). Specifically, Cdc14 function is essential in late M phase: cells carrying a defective mutation arrest in telophase (13), whereas overexpression of Cdc14 results in G1 arrest (12). Cdc14 triggers mitotic cyclin-dependent kinase (CDK)¹ inactivation, enabling cells to exit mitosis through dephosphorylation and activation of the inhibitors of CDKs. At interphase, Cdc14 is a subunit of the mitotic exit network (14–17), which usually localizes to the nucleolus. However, the Cdc14 early anaphase release network initiates the release of Cdc14 from its inhibitor, Net1/Cfi1 (18), and the mitotic exit network promotes further release of Cdc14 from its inhibitor, allowing it to spread into the nucleus and cytoplasm, where it dephosphorylates its major targets (8, 9), leading to exit from mitosis. In addition to this essential role in late M phrase, Cdc14 substrates have also been identified in other stages of the cell cycle (19).Cdc14 putatively regulates 27 proteins (19–22). Some studies have documented the substrates of Cdc14 via in vitro phosphatase assay, whereas others have provided in vivo evidence. However, dephosphorylation sites have been identified for only five of the target proteins (17, 22–25), suggesting that spurious relationships cannot be ruled out. Also, experiments have not been carried out to demonstrate whether these modifications entail direct or indirect regulation. Therefore, our understanding of Cdc14 function and regulation during mitosis in metazoans is incomplete. Conceivably, Cdc14 may regulate many more substrates involved in aspects of chromosome condensation and cytokinesis. To examine this possibility we performed a systematic phosphoproteomic screen to identify new in vivo pathways regulated by Cdc14. Using this approach, we identified both known and potentially novel substrates of Cdc14, as well as their dephosphorylation sites. Many potentially novel substrates are physically associated with Cdc14 in public databases. We also provide biochemical evidence for direct dephosphorylation of the substrates, characterize the specificity of dephosphorylation in two substrates, Smc4 and Bud3, and further study their regulation and critical role in mitosis and cytokinesis.     

New Insights into How the Rho Guanine Nucleotide Dissociation Inhibitor Regulates the Interaction of Cdc42 with Membranes

The subcellular localization of the Rho family GTPases is of fundamental importance to their proper functioning in cells. The Rho guanine nucleotide dissociation inhibitor (RhoGDI) plays a key regulatory role by influencing the cellular localization of Rho GTPases and is essential for the transforming activity of oncogenic forms of Cdc42. However, the mechanism by which RhoGDI helps Cdc42 to undergo the transition between a membrane-associated protein and a soluble (cytosolic) species has been poorly understood. Here, we examine how RhoGDI influences the binding of Cdc42 to lipid bilayers. Despite having similar affinities for the signaling-inactive (GDP-bound) and signaling-active (GTP-bound) forms of Cdc42 in solution, we show that when RhoGDI interacts with Cdc42 along the membrane surface, it has a much higher affinity for GDP-bound Cdc42 compared with its GTP-bound counterpart. Interestingly, the rate for the dissociation of Cdc42·RhoGDI complexes from membranes is unaffected by the nucleotide-bound state of Cdc42. Moreover, the membrane release of Cdc42·RhoGDI complexes occurs at a similar rate as the release of Cdc42 alone, with the major effect of RhoGDI being to impede the re-association of Cdc42 with membranes. These findings lead us to propose a new model for how RhoGDI influences the ability of Cdc42 to move between membranes and the cytosol, which highlights the role of the membrane in helping RhoGDI to distinguish between the GDP- and GTP-bound forms of Cdc42 and holds important implications for how it functions as a key regulator of the cellular localization and signaling activities of this GTPase.The Rho family GTPases are a tightly regulated class of signaling proteins that controls a number of important cellular processes. Known most prominently for their ability to remodel the actin cytoskeleton in mammalian cells (1–3), members of this GTPase family have been shown to play essential roles in cell migration, epithelial cell polarization, phagocytosis, and cell cycle progression (4–11). The Rho family member Cdc42 was discovered for its essential role in bud formation in Saccharomyces cerevisiae (12). However, after its identification in higher organisms (13), Cdc42 has been implicated in a diverse array of signaling pathways including those involved in the regulation of cell growth and in the induction of malignant transformation (14). Indeed, point mutations which enable Cdc42 to undergo the spontaneous exchange of GDP for GTP cause NIH3T3 cells to form colonies in soft agar and grow in low serum, two hallmarks of cellular transformation (15). The introduction of activated Cdc42 mutants into nude mice gives rise to tumor formation (16). Moreover, cellular transformation by oncogenic Ras, one of the most commonly mutated proteins in human cancers, requires the activation of Cdc42 (17).At the molecular level, there are a number of mechanisms that possibly contribute to the roles played by Cdc42 in cell growth control and cellular transformation. These include the ability of Cdc42 to activate the c-Jun NH₂-terminal kinase and p38/Mpk2 signaling pathways (18–20) as well as spatially regulate proteins implicated in the establishment of microtubule-dependent cell polarity including glycogen synthase kinase-3β and adenomatous polyposis coli (21), extend the lifetime of epidermal growth factor receptor-signaling activities by sequestering Cbl, a ubiquitin E3 ligase (22), and influence intracellular trafficking events (23, 24). To mediate such a wide range of cellular responses, two parameters must be properly regulated; that is, the activation state of Cdc42 and its subcellular localization. As is the case with other GTPases, the activation of Cdc42 occurs as an outcome of GDP-GTP exchange, which then enables it to undergo high affinity interactions with effector proteins (25–27). Upon the hydrolysis of GTP to GDP, Cdc42 is converted back to a signaling-inactive state. Two families of proteins work in opposing fashion to regulate the GTP-binding/GTPase cycle of Cdc42. GTPase-activating proteins recognize the GTP-bound form of Cdc42 and accelerate the hydrolysis of GTP to GDP, rendering Cdc42 inactive (28, 29). Guanine nucleotide exchange factors (GEFs)² stimulate the dissociation of GDP from Cdc42, thereby promoting the formation of its signaling-active, GTP-bound state (29, 30).Of equal importance to its activation status is the spatial regulation of Cdc42. This is highly contingent on the particular cellular membranes that serve as sites of binding and/or recruitment of Cdc42 (31–33). The vast majority of in vitro studies performed on Cdc42 have been carried out in the absence of lipids, which is an important omission considering that virtually all of the physiological functions of Cdc42 occur on a membrane surface (34). Cdc42, along with most other Rho family GTPases, undergoes a series of carboxyl-terminal modifications which result in the covalent attachment of a 20-carbon geranylgeranyl lipid anchor (35–37). Directly preceding this lipid tail is a sequence of basic residues that further stabilizes the association of Cdc42 with the membrane surface (31, 33, 38). A ubiquitously expressed 22-kDa protein called Rho guanine nucleotide dissociation inhibitor (RhoGDI) was found to form a soluble (cytosolic) complex with Cdc42 and other Rho GTPases and to apparently promote their release from membranes (39, 40). RhoGDI was originally discovered and named for its ability to block the GEF- and EDTA-stimulated nucleotide exchange activity of Rho family GTPases (39, 41, 42) and then subsequently shown to inhibit the GTP-hydrolytic activity of Cdc42 (43) and to be capable of interacting with the GDP- and GTP-bound forms of Cdc42 in solution with equal affinity (44). The x-ray crystal structure of a complex between RhoGDI and Cdc42-GDP revealed two types of binding interactions (45). An amino-terminal regulatory arm of RhoGDI was shown to form a helix-loop-helix motif that binds to both of the switch domains of Cdc42, leading to the inhibition of GTP hydrolysis and GDP dissociation (45, 46). The carboxyl-terminal two-thirds of RhoGDI assumes an immunoglobulin-like domain, forming a hydrophobic pocket that in effect provides a membrane substitute for the geranylgeranyl moiety of Cdc42. After release from membranes, the lipid anchor of Cdc42 binds in the hydrophobic pocket of RhoGDI, thereby helping to maintain Cdc42 in solution (45–47).Prior work from our laboratory has demonstrated an essential role for RhoGDI in Cdc42-mediated cellular transformation. Based on the x-ray crystal structure for the Cdc42·RhoGDI complex, Arg-66 of Cdc42 makes multiple contacts with RhoGDI. When this residue was changed to alanine, Cdc42 was unable to bind to RhoGDI but was still capable of interacting with its other regulatory and effector proteins. Interestingly, when the R66A mutant of Cdc42 was examined in the constitutively active Cdc42(F28L) background, the resulting Cdc42 double mutant was no longer able to transform cells (48). Knocking down RhoGDI by small interfering RNA also blocked transformation by Cdc42. These findings highlighted a key role for RhoGDI in the ability of Cdc42 to stimulate signaling pathways of importance to cellular transformation, presumably by influencing the membrane association of Cdc42 and ensuring its proper cellular localization.In the present study we have set out to better understand how RhoGDI regulates the signaling functions of Cdc42 and, in particular, how RhoGDI affects the association of Cdc42 with membranes. We show how the membrane plays a previously unappreciated role in allowing RhoGDI to distinguish between the signaling-inactive (GDP-bound) and signaling-active (GTP-bound) forms of Cdc42. By assaying the binding of Cdc42 to insect cell membranes and compositionally defined liposomes through different approaches including a sensitive, real-time fluorescence resonance energy transfer (FRET) readout, we have been able to establish how RhoGDI influences the ability of Cdc42 to transition between a membrane-bound and soluble species. This has led us to propose a new mechanism describing how RhoGDI performs its important regulatory function.     

PRC1 Cooperates with CLASP1 to Organize Central Spindle Plasticity in Mitosis

During cell division, chromosome segregation is governed by the interaction of spindle microtubules with the kinetochore. A dramatic remodeling of interpolar microtubules into an organized central spindle between the separating chromatids is required for the initiation and execution of cytokinesis. Central spindle organization requires mitotic kinesins, microtubule-bundling protein PRC1, and Aurora B kinase complex. However, the precise role of PRC1 in central spindle organization has remained elusive. Here we show that PRC1 recruits CLASP1 to the central spindle at early anaphase onset. CLASP1 belongs to a conserved microtubule-binding protein family that mediates the stabilization of overlapping microtubules of the central spindle. PRC1 physically interacts with CLASP1 and specifies its localization to the central spindle. Repression of CLASP1 leads to sister-chromatid bridges and depolymerization of spindle midzone microtubules. Disruption of PRC1-CLASP1 interaction by a membrane-permeable peptide abrogates accurate chromosome segregation, resulting in sister chromatid bridges. These findings reveal a key role for the PRC1-CLASP1 interaction in achieving a stable anti-parallel microtubule organization essential for faithful chromosome segregation. We propose that PRC1 forms a link between stabilization of CLASP1 association with central spindle microtubules and anti-parallel microtubule elongation.To ensure that each daughter cell receives the full complement of the genome in each cell division, chromosomes move poleward, and non-kinetochore fibers become bundled at the onset of anaphase, initiating assembly of the central spindle, a set of anti-parallel microtubules that serves to concentrate key regulators of cytokinesis (1–3). Chromosomal passengers are a group of evolutionarily conserved proteins that orchestrates chromosome segregation and central spindle plasticity (4, 5). This protein complex containing Aurora B, Survivin, INCENP, and Borealin is relocated from the kinetochore to the central spindle upon anaphase onset (5–9). Perturbation of their function results in defects in metaphase chromosome alignment, chromosome segregation, and cytokinesis (10).Among the central spindle maintenance components, only two have been reported to mediate the microtubule bundling in the central spindle. One is centralspindlin, a heterotetramer containing CeMKLP1/ZEN-4 and RhoGAP/CYK-4 (11), and the other one is an evolutionarily conserved protein, PRC1 (also named Feo in fruit fry, Ase1 in yeast, and MAP65 in plant cells). PRC1 is a non-motor microtubule-binding and -bundling protein in human cells originally identified as a Cdc2 substrate essential for cytokinesis (12, 13). Similar microtubule regulatory activities have been reported in yeast, fruit fly, and plant cells. It is well known that overexpression of wild type PRC1 in HeLa cells can result in thick microtubule bundles in cells at interphase (13). Bundling activity of PRC1, as well as centralspindlin, is required for the organization of the central spindle as well as for the successful progression of cytokinesis. PRC1 molecules accumulate on the midline of a central spindle with the cell cycle progression to anaphase. As a non-motor microtubule-binding protein, transportation of PRC1 to the midline is promoted by its association to kinesin, KIF4A, and timing of this progression is controlled by the dephosphorylation of Thr-481 on PRC1 when the cell exits metaphase by phosphatase Cdc14 (14). Our recent study shows that prevention of the phosphorylation of PRC1 at Thr-470 causes an inhibition in PRC1 oligomerization in vitro and an aberrant organization of central spindle in vivo, suggesting that this phosphorylation-dependent PRC1 oligomerization ensures that central spindle assembly occurs at the appropriate time in the cell cycle (15).Spatiotemporal regulation of microtubule organization and dynamics is responsible for the mitotic apparatus such as the central spindle. However, it has remained elusive as to how the central spindle microtubule organization and dynamics are regulated. There are large varieties of microtubule-associated proteins responsible for regulation of the dynamic behavior of microtubules and microtubule-mediated transport. Among these, proteins that associate with the tips of microtubules are called +TIPs, for “plus-end tracking proteins.” These proteins have been shown to be important in different organisms and cellular systems (16). Using yeast two-hybrid assay, CLASPs were identified as interacting partners of the CLIPs and characterized as new +TIP proteins (17).The microtubule-binding protein CLASP is emerging as an important microtubule regulator in the formation of the mitotic apparatus (18–22). CLASP is required for promoting plus-end growth of spindle microtubules in prometaphase (23). Although the molecular mechanisms underlying its regulation of microtubule dynamics remain elusive, it is generally believed that CLASP orchestrates microtubule dynamics via its physical interacting with EB1, CLIP170, and microtubules (17, 24).To delineate the molecular function of PRC1 in central spindle organization and spatiotemporal regulation, we carried out a new search for PRC1-interacting proteins. Our studies show that PRC1 physically interacts with CLASP1, and the two proteins cooperate in the organization of the central spindle. Our studies provide a novel regulatory mechanism in which the PRC1 complex operates central spindle organization in mitosis.     

Mitotic Regulation of the Stability of Microtubule Plus-end Tracking Protein EB3 by Ubiquitin Ligase SIAH-1 and Aurora Mitotic Kinases

Microtubule plus-end tracking proteins (+TIPs) control microtubule dynamics in fundamental processes such as cell cycle, intracellular transport, and cell motility, but how +TIPs are regulated during mitosis remains largely unclear. Here we show that the endogenous end-binding protein family EB3 is stable during mitosis, facilitates cell cycle progression at prometaphase, and then is down-regulated during the transition to G₁ phase. The ubiquitin-protein isopeptide ligase SIAH-1 facilitates EB3 polyubiquitination and subsequent proteasome-mediated degradation, whereas SIAH-1 knockdown increases EB3 stability and steady-state levels. Two mitotic kinases, Aurora-A and Aurora-B, phosphorylate endogenous EB3 at Ser-176, and the phosphorylation triggers disruption of the EB3-SIAH-1 complex, resulting in EB3 stabilization during mitosis. Our results provide new insight into a regulatory mechanism of +TIPs in cell cycle transition.Microtubule dynamics are essential in many cellular processes, including cell motility, intracellular transport, accurate mitosis, and cytokinesis in all eukaryotes. The regulatory factors for microtubule dynamics can be classified into two main types as follows: microtubule-destabilizing proteins, such as stathmin/Op18 (1) and the Kinesin-13 family (also known as MCAK/KIF2 family) (2), and microtubule-stabilizing proteins, the classic superfamily of microtubule-associated proteins (3). Additionally, the plus-end tracking proteins (+TIPs)³ have recently been identified; this family specifically accumulates at the ends of growing microtubules and regulates the microtubule plus-end targeting to the cell cortex or mitotic kinetochores (4, 5).The EB1 family is a member of the +TIPs family and consists of three homologs in mammals: EB1, EB2/RP1 (henceforth, EB2), and EB3 (6). As EB1 was originally identified as a protein that interacts with the well characterized tumor suppressor adenomatous polyposis coli (APC) protein (7), the function of EB1 has been investigated extensively. EB1 interacts with other +TIPs, including APC, p150^glued, CLIPs, and CLASP1/2, and the interaction network controls microtubule orientation and microtubule-cortex interaction during cell migration (5, 8, 9). EB1 functions not only in the regulation of interphase microtubule dynamics but also in mitotic spindle regulation. For accurate chromosomal segregation, sister chromatids become aligned to the metaphase plate during metaphase, and the alignment requires spindle-kinetochore attachment. Two models have been proposed; in the first, termed the “search-and-capture” model, EB1 localized at the growing microtubule plus-ends searches for binding partners located on kinetochores (10, 11). In the second model proposed recently, EB1 makes kinetochore fibers and centrosomal microtubules connect, and it is essential for the formation of a functional bipolar spindle (12). Thus, EB1 is thought to be a master controller of microtubule plus-ends; however, little is known about other EB1 family members. Given that EB3 is localized on the microtubule network and binds to APC and CLIPs identically to EB1, it is possible that EB3 acts as an EB1 analog in cells (13–15).Cell division is precisely regulated by several post-translational modifications of proteins, mainly reversible phosphorylation and ubiquitination, which is followed by degradation. Accurate mitotic phase progression requires the appropriate phosphorylation of various proteins by mitotic kinases (16, 17). One of the key mitotic kinases is the Aurora family that has been highly conserved from yeast to humans. There are three homologs (Aurora-A, -B, and -C) in human and mouse (18). Although their homology at the protein level is more than 84%, their functions and subcellular localizations are distinct. Aurora-A is located in the centrosomes and spindle and is required for mitotic entry, centrosome maturation/separation, and spindle assembly (19). Aurora-B is a chromosomal passenger protein that localizes on the inner centromere of the chromosomes until metaphase to regulate the spindle-kinetochore attachment, and from anaphase, it translocates to the central spindle and then accumulates in the midbody for cytokinesis (20, 21). The numerous substrates of the Aurora family include regulatory factors for microtubule dynamics, such as the microtubule-destabilizing proteins MCAK and stathmin, which help to establish the bipolar attachment and spindle assembly, respectively (22–24). It is possible that the Aurora family regulates the EB1 family by phosphorylation.In this study, we performed yeast two-hybrid screening and obtained the EB1 yeast homolog Bim1 as a protein that interacts with Ipl1, a yeast counterpart of Aurora. Here we demonstrate the novel regulatory mechanisms of EB3 by two cell cycle-dependent post-translational modifications, phosphorylation and ubiquitin-proteasome-mediated degradation.     

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.Oligomeric heat shock protein 27 (Hsp27)² is a ubiquitous mammalian protein with a variety of functions in health and disease (1–8). These functions include ATP-independent chaperone activity in response to environmental stress, e.g. heat shock and oxidative stress, control of apoptosis, and regulation of actin cytoskeleton dynamics. Hsp27 is a member of the α-crystallin small heat shock protein family of which αB-crystallin is the archetype. These proteins are characterized by an α-crystallin domain of 80–90 residues consisting of roughly eight β-strands that form an intermolecular β-sheet interaction interface within a dimer, the basic building subunit of the oligomer (2, 4, 9–11).Hsp27 is in equilibrium between high molecular weight oligomers and much lower molecular weight multimers. It has been reported that unphosphorylated Hsp27 includes predominantly a distribution of high molecular species ranging in size from 12-mer to 35-mer (12–19). Phosphorylation of Hsp27 at serines 15, 78, and 82 by the p38-activated MAPKAP-2 kinase (20–22) or the use of the triple Ser-to-Asp phospho-mimic results in a major shift in the equilibrium toward much smaller multimers (23) and in an alteration of its function (1, 3, 6, 7, 24, 25). The size distribution of the smaller species has been reported to be between monomer and tetramer (12–16, 18, 19).Small heat shock proteins, including Hsp27, behave as ATP-independent molecular chaperones during cellular heat shock. They bind partially unfolded proteins and prevent their aggregation until the proteins can be refolded by larger ATP-dependent chaperones or are digested (7, 8, 26). This function includes the up-regulation and/or phosphorylation of Hsp27.It is not entirely clear what the role of Hsp27 size and phosphorylation state plays in its heat shock function because there are conflicting results in the literature. Some in vitro studies concluded that the unphosphorylated oligomeric Hsp27 (or the murine isoform Hsp25) protects proteins against aggregation better than does the phosphorylation mimic (13, 19, 27), whereas others found no difference (16, 28, 29), and still other studies found that the mimic protects better than does the unphosphorylated wild type (27, 30, 31). In-cell studies found that phosphorylation of Hsp27 was essential for thermo-protection of actin filaments (32), and the Hsp27 phosphorylation mimic decreased inclusion body formation better than did unphosphorylated Hsp27 (33). This study was undertaken to investigate the molecular chaperone function of Hsp27 by correlating chaperone activity with Hsp27 size and by comparing fully phosphorylated Hsp27 with its phospho-mimic.     

Cdk1-Cyclin B1-mediated Phosphorylation of Tumor-associated Microtubule-associated Protein/Cytoskeleton-associated Protein 2 in Mitosis

During mitosis, establishment of structurally and functionally sound bipolar spindles is necessary for maintaining the fidelity of chromosome segregation. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a mitotic spindle-associated protein whose level is frequently up-regulated in various malignancies. Previous reports have suggested that TMAP is a potential regulator of mitotic spindle assembly and dynamics and that it is required for chromosome segregation to occur properly. So far, there have been no reports on how its mitosis-related functions are regulated. Here, we report that TMAP is hyper-phosphorylated at the C terminus specifically during mitosis. At least four different residues (Thr-578, Thr-596, Thr-622, and Ser-627) were responsible for the mitosis-specific phosphorylation of TMAP. Among these, Thr-622 was specifically phosphorylated by Cdk1-cyclin B1 both in vitro and in vivo. Interestingly, compared with the wild type, a phosphorylation-deficient mutant form of TMAP, in which Thr-622 had been replaced with an alanine (T622A), induced a significant increase in the frequency of metaphase cells with abnormal bipolar spindles, which often displayed disorganized, asymmetrical, or narrow and elongated morphologies. Formation of these abnormal bipolar spindles subsequently resulted in misalignment of metaphase chromosomes and ultimately caused a delay in the entry into anaphase. Moreover, such defects resulting from the T622A mutation were associated with a decrease in the rate of protein turnover at spindle microtubules. These findings suggest that Cdk1-cyclin B1-mediated phosphorylation of TMAP is important for and contributes to proper regulation of microtubule dynamics and establishment of functional bipolar spindles during mitosis.Tumor-associated microtubule-associated protein (TMAP),³ also known as cytoskeleton-associated protein 2 (CKAP2), LB-1, and se20-10, is frequently up-regulated in various malignancies, including gastric adenocarcinoma, diffuse B-cell lymphoma, and cutaneous T-cell lymphoma (1–3), and detected in various cancer cell lines (1, 4). Knockdown of TMAP significantly reduces the rate of cell growth (5, 6), indicating that it is essential for normal cell growth. However, the cellular functions of TMAP remain largely unknown. Recent findings indicate that TMAP plays an essential role in mitosis. Expression of TMAP changes in a cell cycle-dependent manner; its expression is relatively low during G₁, starts to incline during G₁/S transition, and peaks at G₂/M phases of the cell cycle (5, 7). TMAP primarily localizes at mitotic spindle and spindle poles during mitosis (1, 4, 8, 9). During late stages of mitosis, however, TMAP localizes near the chromatin region and to the midbody microtubules (8). TMAP has microtubule-stabilizing properties (4, 8, 9), and its overexpression induces mitotic spindle defects, including monopolar spindle formation, and arrests cells at mitosis as a result (8). Similar to other mitotic regulators, TMAP is a substrate of the anaphase-promoting complex (8). TMAP is degraded during mitotic exit by the anaphase-promoting complex-Cdh1 in a KEN box-dependent manner. Results of the experiments using a nondegradable mutant of TMAP suggested that proper regulation of the TMAP protein level is functionally important for establishment of bipolar spindles and completion of cytokinesis. Recently, we also have shown that siRNA-mediated depletion of TMAP in mammalian cells results in chromosome missegregation, characterized by chromatin bridge formation and malformation of interphase nuclei, and such phenotype was associated with a reduction in the spindle assembly checkpoint activity (6). These findings suggest that TMAP is a potential regulator of mitotic spindle function and dynamics and that proper regulation of its protein level and functions is necessary for establishment of bipolar spindles as well as for maintaining the fidelity of the chromosome segregation process.At the onset of mitosis, the microtubule network undergoes extensive rearrangements to form a unique bipolar structure, called the mitotic spindle. Multiple factors have been shown to associate with the mitotic spindle and regulate its function by influencing its assembly and dynamics (10, 11). Establishment of a functional bipolar mitotic spindle is critical for faithful segregation of sister chromatids and maintenance of genomic stability. In support of this notion, disruption or depletion of factors involved in regulation of the spindle microtubule dynamics or establishment of spindle bipolarity have been shown to induce spindle dysfunction and ultimately chromosome missegregation (12–14).The cyclin-dependent kinase 1 (Cdk1) in complex with cyclin B1 (Cdk1-cyclin B1) is one of the key mitotic kinases. The kinase activity of Cdk1-cyclin B1 governs the entry into mitosis from G₂ phase of the cell cycle (15, 16). Through mediating phosphorylation of a variety of substrates, Cdk1-cyclin B1 also plays an important role in multiple processes during mitosis, including chromosome condensation, nuclear envelope breakdown, centrosome separation, regulation of spindle microtubule dynamics, and metaphase to anaphase transition (17–20). In particular, a number of regulators of microtubules are among Cdk1-cyclin B1 substrates (21). For instance, phosphorylation of a kinesin-related motor protein, Eg5, by Cdk1-cyclin B1 is necessary for its centrosomal localization and ultimately for the centrosome separation process to occur properly (18). Also, Cdk1-cyclin B1-mediated phosphorylation of some of the effectors of microtubule dynamics has been shown to regulate their microtubule-stabilizing or -destabilizing activities during mitosis (22, 23). These suggest that the assembly and maintenance of bipolar spindles during mitosis are under regulation of Cdk1-cyclin B1.We have recently reported that TMAP is phosphorylated specifically during mitosis (24), which led us to hypothesize that the mitotic functions of TMAP are regulated by timely phosphorylation. In the present study, we identified multiple, mitosis-specific phosphorylation sites on TMAP, one of which is phosphorylated by Cdk1-cyclin B1, and investigated the functional importance of Cdk1-cyclin B1-mediated phosphorylation of TMAP during mitosis.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]