Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18

The way that microtubules reorganize from their long, stable interphase configuration to form the mitotic spindle remains a challenging and unsolved question. It is now widely recognized that microtubule polymerization during the cell cycle is regulated by a balance between microtubule-stabilizing and-destabilizing factors. Stabilizing factors include a large group of microtubule-associated proteins (MAPs; e.g. MAP4, XMAP215, XMAP230/XMAP4 and XMAP310) and the destabilizing factors are a growing family of proteins (e.g. Stathmin/Op18 and XKCM1). Recent studies have allowed a mechanistic dissection of how these stabilizing and destabilizing factors regulate microtubule dynamics and spindle assembly.     

Stathmin/Op18 is a novel mediator of vinblastine activity

Microtubule (MT) dynamic instability is tightly regulated by stabilizing and destabilizing proteins, the latter being exemplified by stathmin/Op18, a protein known to destabilize MTs. Studies in cells have indicated that the level of stathmin expression modifies the cytotoxicity of antimicrotubule drugs, such as vinblastine (VLB). Using isothermal titration calorimetry and analytical ultracentrifugation, we show that VLB increases the affinity of stathmin for tubulin 50-fold (and vice versa). These results are the first biochemical evidence of the direct relationship between stathmin and an antimitotic drug, and reveal a new mechanism of action for VLB.     

Stathmin/Oncoprotein 18(Op18)基因在人骨肉瘤中的表达及其意义

目的:探讨stathmin/oncoprotein 18(Op18)基因在人骨肉瘤中的表达规律及意义。方法:分别采用免疫组化法和Western blot法检测10例正常骨组织和56例骨肉瘤中stathmin蛋白的表达。结果:免疫组化法检测stathmin蛋白在正常骨组织、低级别骨肉瘤(G1级组)及高级别骨肉瘤(G2级组)中阳性表达率分别为20%、65%、100%。正常骨组织分别与G1级组、G2级组比较,均有显著性差异(P﹤0.05);G1级组与G2级组比较,有显著差异(P<0.05)。Western blot法检测显示,stathmin蛋白在正常骨组织、G1级组、G2级组表达相对值分别为(0.34±0.15)、(0.68±0.21)、(0.90±0.17)。正常骨组织分别与G1级级组、G2级组比较,差异均有显著性(P<0.01),G1级组与G2级组比较,差异有显著性(P<0.01)。结论:Stathmin在骨肉瘤中过表达,与骨肉瘤的发生及发展密切相关。     

Deciphering the cellular functions of the Op18/Stathmin family of microtubule-regulators by plasma membrane-targeted localization

The Op18/stathmin family of microtubule regulators includes the ubiquitous cytosolic Op18/stathmin (Op18) and the neuronal, primarily Golgi-associated proteins SCG10 and RB3, which all form ternary complexes with two head-to-tail-aligned tubulin heterodimers. To understand the physiological significance of previously observed differences in ternary complex stability, we have fused each of the heterodimer-binding regions of these three proteins with the CD2 cell surface protein to generate confined plasma membrane localization of the resulting CD2 chimeras. Herein, we show that, in contrast to constitutively active CD2-Op18-tetraA, both the CD2-SCG10 and CD2-RB3 chimeras sequestered tubulin at the plasma membrane, which results in >35% reduction of cytosolic tubulin heterodimer levels and consequent delayed formation of mitotic spindles. However, all three CD2 chimeras, including the tubulin sequestration-incompetent CD2-Op18-tetraA, destabilize interphase microtubules. Given that microtubules are in extensive contact with the plasma membrane during interphase, but not during mitosis, these findings indicate that Op18-like proteins have the potential to destabilize microtubules by both sequestration and direct interaction with microtubules. However, the differences in tubulin binding observed in cells also indicate conceptual differences between the functions of low-abundance neural family members, which will accumulate tubulin at specific cellular compartments, and the abundant cytosolic Op18 protein, which will not.     

Op18/Stathmin counteracts the activity of overexpressed tubulin-disrupting proteins in a human leukemia cell line

Op18/stathmin (Op18) is a phosphorylation-regulated and differentially expressed microtubule-destabilizing protein in animal cells. Op18 regulates tubulin monomer-polymer partitioning of the interphase microtubule system and forms complexes with tubulin heterodimers. Recent reports have shown that specific tubulin-folding cofactors and related proteins may disrupt tubulin heterodimers. We therefore investigated whether Op18 protects unpolymerized tubulin from such disruptive activities. Our approach was based on inducible overexpression of two tubulin-disrupting proteins, namely TBCE, which is required for tubulin biogenesis, and E-like, which has been proposed to regulate tubulin turnover and microtubule stability. Expression of either of these proteins was found to cause a rapid degradation of both alpha-tubulin and beta-tubulin subunits of unpolymerized, but not polymeric, tubulin heterodimers. We found that depletion of Op18 by means of RNA interference increased the susceptibility of tubulin to TBCE or E-like mediated disruption, while overexpressed Op18 exerted a tubulin-protective effect. Tubulin protection was shown to depend on Op18 levels, binding affinity, and the partitioning between tubulin monomers and polymers. Hence, the present study reveals that Op18 at physiologically relevant levels functions to preserve the integrity of tubulin heterodimers, which may serve to regulate tubulin turnover rates.     

Regulation of microtubule destabilizing activity of Op18/stathmin downstream of Rac1

In the leading edge of migrating cells, a subset of microtubules exhibits net growth in a Rac1- and p21-activated kinase-dependent manner. Here, we explore the possibility of whether phosphorylation and inactivation of the microtubule-destabilizing protein Op18/stathmin could be a mechanism regulating microtubule dynamics downstream of Rac1 and p21-activated kinases. We find that, in vitro, Pak1 phosphorylates Op18/stathmin specifically at serine 16 and inactivates its catastrophe promoting activity in biochemical and time lapse microscopy microtubule assembly assays. Furthermore, phosphorylation of either serine 16 or 63 is sufficient to inhibit Op18/stathmin in vitro. In cells, the microtubule-destabilizing effect of an excess of Op18/stathmin can be partially overcome by expression of constitutively active Rac1(Q61L), which is dependent on Pak activity, suggesting that the microtubule cytoskeleton can be regulated through inactivation of Op18/stathmin downstream of Rac1 and Pak in vivo. However, in vivo, Pak1 activity alone is not sufficient to phosphorylate Op18, indicating that additional pathways downstream of Rac1 are required for Op18 regulation.     

Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3-XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A-dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.     

Cell cycle progression is associated with distinct patterns of phosphorylation of Op18.

Op18 is a highly conserved major cytosolic phosphoprotein which has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. Following mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study we have characterized Op18 phosphorylation during progression of freshly isolated PBL through the cell cycle. Transition from G0 to G1 following activation with OKT3 was associated with an increase in a phosphorylated form designated Op18c. Progression of cells through G1 into S resulted in an increase in phosphorylated Op18 forms, designated Op18a and Op18b, which paralleled new Op18 synthesis. Transition of cells into G2 + M resulted in the appearance of the more acidic phosphorylated forms Op18d and Op18e. Calphostin C, a specific inhibitor of protein kinase C, dramatically decreased all forms of phosphorylated Op18 in OKT3 treated Jurkat cells. Our results suggest that Op18 phosphorylation is mediated in part by PKC activation as well as by other kinases yielding different phosphorylated forms at specific stages of the cell cycle.     

Unidirectional microtubule assembly in cell-free extracts of Spisula solidissima oocytes is regulated by subtle changes in pH

Most, if not all, microtubules in vivo grow unidirectionally from a nucleation site such as the centrosome. This organized growth of microtubules can generate and maintain the radially symmetrical array of interphase microtubules as well as the bipolar mitotic apparatus. To investigate the regulation of polarized microtubule growth, we have prepared a cell-free extract from surf clam oocytes that exhibits unidirectional microtubule assembly. Immunofluorescence microscopy was used to visualize the net assembly of microtubules onto the fast (plus)- and slow (minus)- growing ends of isolated ciliary axonemes. All detectable microtubule growth in these cytoplasmic extracts occurred at the plus (+) ends and the extent of (+) end growth was regulated by subtle changes in pH. Microtubule assembly in these crude extracts was highly favored at pH 7.3, the pH of the post-fertilization cytoplasm. In contrast, when tubulin was purified from these oocyte extracts, integral components were lost, and microtubule growth became predominantly bidirectional and was favored at acidic pH. These results indicate that cytoplasmic factors may inhibit bidirectional growth in vivo and that temporal or local changes in cytoplasmic pH may influence microtubule assembly during the cell cycle.     

Exocyst function is regulated by effector phosphorylation

The exocyst complex tethers vesicles at sites of fusion through interactions with small GTPases. The G protein RalA resides on Glut4 vesicles, and binds to the exocyst after activation by insulin, but must then disengage to ensure continuous exocytosis. Here we report that, after recognition of the exocyst by activated RalA, disengagement occurs through phosphorylation of its effector Sec5, rather than RalA inactivation. Sec5 undergoes phosphorylation in the G-protein binding domain, allosterically reducing RalA interaction. The phosphorylation event is catalysed by protein kinase C and is reversed by an exocyst-associated phosphatase. Introduction of Sec5 bearing mutations of the phosphorylation site to either alanine or aspartate disrupts insulin-stimulated Glut4 exocytosis, as well as other trafficking processes in polarized epithelial cells and during development of zebrafish embryos. The exocyst thus serves as a 'gatekeeper' for exocytic vesicles through a circuit of engagement, disengagement and re-engagement with G proteins.     

APP processing is regulated by cytoplasmic phosphorylation

Amyloid-beta peptide (Abeta) aggregate in senile plaque is a key characteristic of Alzheimer's disease (AD). Here, we show that phosphorylation of amyloid precursor protein (APP) on threonine 668 (P-APP) may play a role in APP metabolism. In AD brains, P-APP accumulates in large vesicular structures in afflicted hippocampal pyramidal neurons that costain with antibodies against endosome markers and the beta-secretase, BACE1. Western blot analysis reveals increased levels of T668-phosphorylated APP COOH-terminal fragments in hippocampal lysates from many AD but not control subjects. Importantly, P-APP cofractionates with endosome markers and BACE1 in an iodixanol gradient and displays extensive colocalization with BACE1 in rat primary cortical neurons. Furthermore, APP COOH-terminal fragments generated by BACE1 are preferentially phosphorylated on T668 verses those produced by alpha-secretase. The production of Abeta is significantly reduced when phosphorylation of T668 is either abolished by mutation or inhibited by T668 kinase inhibitors. Together, these results suggest that T668 phosphorylation may facilitate the BACE1 cleavage of APP to increase Abeta generation.     

Differential effect of two stathmin/Op18 phosphorylation mutants on Xenopus embryo development

Stathmin/Op18 destabilizes microtubules in vitro and regulates microtubule polymerization in vivo. Both a microtubule catastrophe-promoting activity and a tubulin sequestering activity were demonstrated for stathmin in vitro, and both could contribute to microtubule depolymerization in vivo. Stathmin activity can be turned down by extensive phosphorylation on its four phosphorylatable serines, and down-regulation of stathmin activity by phosphorylation is necessary for cells to proceed through mitosis. We show here that microinjection of a nonphosphorylatable Ser to Ala (4A) quadruple mutant in Xenopus two-cell stage embryos results in cell cleavage arrest in the injected blastomeres and aborted development, whereas injection of a pseudo-phosphorylated Ser to Glu quadruple mutant (4E) does not prevent normal development. Addition of these mutants to mitotic cytostatic factor-arrested extracts in which spindle assembly was induced led to a dramatic reduction of spindle size with 4A stathmin, and to a moderate increase with 4E stathmin, but both localized to spindle poles. Interestingly, the microtubule assembly-dependent phosphorylation of endogenous stathmin was abolished in the presence of 4A stathmin, but not of 4E stathmin. Altogether, this shows that the phosphorylation-mediated regulation of stathmin activity during the cell cycle is essential for early Xenopus embryonic development.     

The translocation of focal adhesion kinase in brain synaptosomes is regulated by phosphorylation and actin assembly

Focal adhesion kinase (FAK) and the related proline-rich tyrosine kinase 2 (PYK2) are non-receptor protein tyrosine kinases that transduce extracellular signals through the activation of Src family kinases and are highly enriched in neurones. To further elucidate the regulation of FAK and PYK2 in nervous tissue, we investigated their distribution in brain subcellular fractions and analysed their translocation between membrane and cytosolic compartments. We have found that FAK and PYK2 are present in a small membrane-associated pool and a larger cytosolic pool in various neuronal compartments including nerve terminals. In intact nerve terminals, inhibition of Src kinases inhibited the membrane association of FAK, but not of PYK2, whereas tyrosine phosphatase inhibition sharply increased the membrane association of both FAK and PYK2. Disruption of the actin cytoskeleton was followed by a decrease in the membrane-associated pool of FAK, but not of PYK2. For both kinases, a significant correlation was found between autophosphorylation and membrane association. The data indicate that FAK and PYK2 are present in nerve terminals and that the membrane association of FAK is regulated by both phosphorylation and actin assembly, whereas that of PKY2 is primarily dependent on its phosphorylation state.     

RhoE function is regulated by ROCK I-mediated phosphorylation

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes. Phosphorylation has no effect on RhoE binding to ROCK I, but instead increases RhoE protein stability. Using phospho-specific antibodies, we show that ROCK phosphorylates endogenous RhoE at serine 11 upon cell stimulation with platelet-derived growth factor, and that this phosphorylation requires an active protein kinase C signalling pathway. In addition, we demonstrate that phosphorylation of RhoE correlates with its activity in inducing stress fibre disruption and inhibiting Ras-induced transformation. This is the first demonstration of an endogenous Rho family member being phosphorylated in vivo and indicates that phosphorylation is an important mechanism to control the stability and function of this GTPase-deficient Rho protein.     

Filamentous smooth muscle myosin is regulated by phosphorylation

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody- stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.     

Long-chain acyl-CoA synthetase 4 is regulated by phosphorylation

Long chain acyl CoA synthetase 4 (Acsl4) is a key enzyme in steroidogenesis. It participates in steroid synthesis through of arachidonic acid release and Steroidogenic Acute Regulatory protein (StAR) induction.Acsl4 prefers arachidonic acid as substrate and acts probably as a homodimer. In steroidogenic cells, it has been demonstrated that Acsl4 is a high turnover protein located mainly in mitochondrial-associated membrane fraction (MAM) bound to other proteins and that it is newly synthesized by hormone stimulation. The synthesis of Acsl4 constitutes an early step in steroidogenesis.In the steroid synthesis process, activation of kinases plays a very important role. For this reason, the aim of this work was to study Acsl4 as a possible phosphoprotein and try to elucidate the role of its phosphorylation.We have determined for the first time that Acsl4 is a phosphoprotein whose phosphorylation is hormone-dependent. We also demonstrated that Acsl4 acts effectively as a dimer and that phosphorylation occurs after dimer formation.Studies in vitro demonstrated that Acsl4 is a substrate of both PKA and PKC and its phosphorylation by these kinases regulates its activity.