Intermediate filament reorganization during mitosis is mediated by p34cdc2 phosphorylation of vimentin

As cells enter mitosis, the intermediate filament (IF) networks of interphase BHK-21 cells are depolymerized to form cytoplasmic aggregates of disassembled IFs, and the constituent IF proteins, vimentin and desmin are hyperphosphorylated at several specific sites. We have characterized one of two endogenous vimentin kinases from a particulate fraction of mitotic cell lysates. Through several purification steps, vimentin kinase activity copurifies with histone H1 kinase and both activities bind to p13suc1-Sepharose. The final enriched kinase preparation consists primarily of p34cdc2 and polypeptides of 65 and 110 kd. The purified kinase complex phosphorylates vimentin in vitro at a subset of sites phosphorylated in vivo during mitosis. Furthermore, phosphorylation of in vitro polymerized vimentin IFs by the purified kinase causes their disassembly. Therefore, vimentin is a substrate of p34cdc2 and phosphorylation of vimentin contributes to M phase reorganization of the IF network.     

p34cdc2 is physically associated with and phosphorylated by a cdc2-specific tyrosine kinase.

The mammalian homologue of the yeast cdc2 gene encodes a 34-kilodalton serine/threonine kinase that is a subunit of M phase-promoting factor. Recent studies have shown that p34cdc2 is also a major tyrosine-phosphorylated protein in HeLa cells and that its phosphotyrosine content is cell cycle regulated and related to its kinase activity. Here, we show that cdc2 is physically associated with and phosphorylated in vitro by a highly specific tyrosine kinase. Tyrosine phosphorylation of cdc2 in vitro occurs at tyrosine 15, the same site that is phosphorylated in vivo. The association between the two kinases takes place in the cytosolic compartment and involves cyclin B-associated cdc2. Evidence is presented that a substantial fraction of cytosolic cdc2 is hypophosphorylated, whereas nuclear cdc2 is hyperphosphorylated. Finally, we show that the tyrosine kinase associated with cdc2 may be a 67-kilodalton protein and is distinct from src, abl, fms, and other previously reported tyrosine kinases.     

Ribosomal protein S3 is phosphorylated by Cdk1/cdc2 during G2/M phase

Ribosomal protein S3 (rpS3) is a multifunctional protein involved in translation, DNA repair, and apoptosis. The relationship between rpS3 and cyclin-dependent kinases (Cdks) involved in cell cycle regulation is not yet known. Here, we show that rpS3 is phosphorylated by Cdk1 in G2/M phase. Co-immunoprecipitation and GST pull-down assays revealed that Cdk1 interacted with rpS3. An in vitro kinase assay showed that Cdk1 phosphorylated rpS3 protein. Phosphorylation of rpS3 increased in nocodazole-arrested mitotic cells; however, treatment with Cdk1 inhibitor or Cdk1 siRNA significantly attenuated this phosphorylation event. The phosphorylation of a mutant form of rpS3, T221A, was significantly reduced compared with wild-type rpS3. Decreased phosphorylation and nuclear accumulation of T221A was much more pronounced in G2/M phase. These results suggest that the phosphorylation of rpS3 by Cdk1 occurs at Thr221 during G2/M phase and, moreover, that this event is important for nuclear accumulation of rpS3.     

Mitosis is swell

Cell volume and dry mass are typically correlated. However, in this issue, Zlotek-Zlotkiewicz et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505056) and Son et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201505058) use new live-cell techniques to show that entry to mitosis coincides with rapid cell swelling, which is reversed before division.How growth is linked to division by the cell cycle regulatory network is an important open question in cell biology (Turner et al., 2012; Ginzberg et al., 2015). Yet, what is meant by cell growth? Different methods have been used to estimate either the total dry mass of the cell, total protein content, or cell volume. Although these parameters are often highly correlated, they are not the same. In budding yeast, growth parameters are nearly interchangeable as cell density changes only about 1% through the division cycle (Bryan et al., 2010). In contrast, cell density can drop by over 50% during a rapid growth phase in hypertrophic chondrocytes, which are responsible for determining bone length (Cooper et al., 2013). However, because of the current lack of similarly dramatic examples, it is assumed that chondrocytes are a special case and that most animal cells also exhibit little variation in cell density, as recently measured (Bryan et al., 2014). Yet this assumption has not been thoroughly tested because of the difficulty of measuring cell volume in animal cells, which are often irregularly shaped.Measuring cell volume is even more challenging in live cells. Whereas there is an accurate live-cell method for measuring dry mass in quantitative phase microscopy (Sung et al., 2013), live-cell volume measurements of adherent cells have been difficult because of their irregular geometry. Current methods are mostly based on 3D geometric reconstructions from confocal sections. However, confocal microscopy has poor resolution in the z-dimension, and increasing the number of z-sections to better estimate the cell membrane location and improve accuracy can be phototoxic.In this issue, Son et al. and Zlotek-Zlotkiewicz et al. applied two different methods to accurately measure cell volume changes in live cells. Son et al. (2015) used a variation of the suspended microchannel resonator pioneered by the Manalis laboratory (Fig. 1 A; Burg et al., 2007). In this method, the resonance frequency of the device shifts when a cell enters a part of a microchannel because the cell is of a different density than the surrounding media. The change in resonance frequency can therefore be used to calculate the buoyant mass of the cell. Changing the media of the microchannel to one of different density and then performing the same measurement for the same cell allows the accurate calculation of both cell dry mass and volume. One limitation of the microchannel resonator method is that the cells are required to be nonadherent so that they can be moved into and out of the resonator. To measure cell volume of adherent cells, Zlotek-Zlotkiewicz et al. (2015) used a microchamber culture device with a low 15–25-µm adjustable ceiling (Fig. 1 B). Cells were grown in a media containing fluorescent dye–labeled dextran. Cell volume could then be measured from epifluorescence images because the cells displaced the fluorescent dextran in proportion to their volume. This method was combined with quantitative phase microscopy to measure dry mass.Open in a separate windowFigure 1.Two new live-cell measurements of cell volume and mass reveal that cells swell in mitosis. (A) Schematic of microchannel resonator whose frequency is determined by the cells’ buoyant mass. Live-cell measurements in two media of different density allow calculation of cell volume and density (modified from Son et al., 2015). (B) Using epifluorescence microscopy, cell volume can be measured as the amount of dye-labeled dextran displaced in a low-ceiling culture chamber. (C) Cell density is constant through the cell cycle except in mitosis, when cells swell (modified from Son et al., 2015). (D) In the context of an animal tissue, mitotic swelling may generate a larger, rounder space to promote accurate and rapid chromosome segregation.Both Son et al. (2015) and Zlotek-Zlotkiewicz et al. (2015) applied their methods to precisely and noninvasively measure the volume and density dynamics in growing and dividing mammalian cells (Fig. 1 C). During most of the cell cycle, density is constant and dry mass is correlated with volume. However, the researchers found that cell volume, but not dry mass, increases rapidly as cells enter mitosis. This osmotic swelling occurs during prophase and prometaphase before being reversed in anaphase and telophase. Collectively, the work of both teams also determined that mitotic swelling is driven by osmotic water exchange and requires the activity of the Na/H ion exchanger but is not dependent on the actomyosin cortex, endocytosis, or cytokinesis. Whereas previous studies gave contradictory results, the two papers in this issue show that there is a reversible 10–30% volume increase during mitosis depending on the type of cell.The establishment of cell swelling during mitosis raises the question of its function. In laboratory conditions, mitotic animal cells lose surface adhesion and are spherical. This spherical geometry is accompanied by an increase in intracellular hydrostatic pressure (Stewart et al., 2011). In the in vivo context of an animal tissue, an increase in intracellular pressure accompanied by cell swelling would allow cells to push against their neighbors and open up additional space for mitosis (Fig. 1 D; Son et al., 2015; Zlotek-Zlotkiewicz et al., 2015). The mitotic acquisition of a larger, more spherical geometry may be important because physically preventing cells from rounding up retards mitosis and promotes inaccurate chromosome segregation (Lancaster et al., 2013). Alternatively, the dilution of the cytoplasm by swelling might change the physicochemical properties of the intracellular environment to facilitate chromosomal movement and segregation or change the kinetics of biochemical reactions (Son et al., 2015).Live-cell methods that accurately measure volume will most obviously be useful for studies of how cell growth is linked to cell cycle progression but are unlikely to be limited to this application. For example, it would be interesting to follow the dynamics of cell volume and density in other processes in which the surface area to volume ratio can change rapidly, such as cell migration (Traynor and Kay, 2007). Depending on the environment, cells can switch from actin-driven motility to hydrostatic pressure–driven bleb-based motility (Sahai and Marshall, 2003; Zatulovskiy et al., 2014). Because this motility switch strongly depends on the osmolarity of the environment (Fedier and Keller, 1997; Yoshida and Soldati, 2006), it is likely to be accompanied by and perhaps even require cell swelling.Although the swelling of animal cells has been mostly neglected, cell swelling is not unusual in other eukaryotic lineages. Unlike animal cells, which have a flexible cell geometry that can rapidly be remodeled, plant and fungal cells have a stiff cell wall and cannot easily change their geometry. Nevertheless, plants and fungi can use regulated swelling to move on time scales faster than that of growth (Skotheim and Mahadevan, 2005). For example, a stem bending to track the sun is caused by cells on one side swelling, whereas those on the other side shrink. This differential swelling allows the stem to bend because the plant tissue is connected by elastic cell walls. Using such differential swelling, plants and fungi can perform impressive coordinated movements to track the sun, compete for territory, disperse seeds or spores, and catch prey (Attenborough, 1995). Although swelling-based movements have long been appreciated in the context of plants, there is no a priori reason animal cells might not also harness such mechanisms to perform important functions. The further development and dissemination of technologies to accurately measure cell volume, density, and dry mass, such as those described in this issue, will be essential to determine the extent to which animal cells harness swelling.     

磷酸化组蛋白H3在小麦有丝分裂与减数分裂中的分布

在细胞周期中 ,与染色质凝集偶联的一类组蛋白修饰是组蛋白H3的磷酸化。运用H3_Ser 10磷酸化的特异性抗体 ,通过间接免疫荧光标记检测了磷酸化组蛋白H3在小麦 (TriticumaestivumL .)有丝分裂与减数分裂细胞中的分布。有丝分裂时 ,H3磷酸化起始于早前期 ,消失于末期 ,在中期与后期 ,H3磷酸化主要分布在着丝粒两侧的异染色质区。减数分裂时 ,H3磷酸化起始于细线期向偶线期转换时 ,并且从前期Ⅰ到后期Ⅰ保持均一分布于整个染色体上 ,直到末期Ⅰ消失 ,而中期Ⅱ与后期Ⅱ在着丝粒两侧的异染色质区的信号略强于染色体臂 ,直至消失于末期Ⅱ。磷酸化组蛋白H3在两类细胞分裂中的不同分布暗示这种保守的翻译后修饰可能发挥着除参与染色体凝集外的更复杂的作用。     

磷酸化组蛋白H3在小麦有丝分裂与减数分裂中的分布

在细胞周期中, 与染色质凝集偶联的一类组蛋白修饰是组蛋白H3的磷酸化.运用H3-Ser 10磷酸化的特异性抗体,通过间接免疫荧光标记检测了磷酸化组蛋白H3在小麦(Triticum aestivum L.)有丝分裂与减数分裂细胞中的分布.有丝分裂时,H3磷酸化起始于早前期,消失于末期,在中期与后期,H3磷酸化主要分布在着丝粒两侧的异染色质区.减数分裂时,H3磷酸化起始于细线期向偶线期转换时,并且从前期Ⅰ到后期Ⅰ保持均一分布于整个染色体上,直到末期Ⅰ消失,而中期Ⅱ与后期Ⅱ在着丝粒两侧的异染色质区的信号略强于染色体臂,直至消失于末期Ⅱ.磷酸化组蛋白H3在两类细胞分裂中的不同分布暗示这种保守的翻译后修饰可能发挥着除参与染色体凝集外的更复杂的作用.     

cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2

cdc25 controls the activity of the cyclin-p34cdc2 complex by regulating the state of tyrosine phosphorylation of p34cdc2. Drosophila cdc25 protein from two different expression systems activates inactive cyclin-p34cdc2 and induces M phase in Xenopus oocytes and egg extracts. We find that the cdc25 sequence shows weak but significant homology to a phylogenetically diverse group of protein tyrosine phosphatases. cdc25 itself is a very specific protein tyrosine phosphatase. Bacterially expressed cdc25 directly dephosphorylates bacterially expressed p34cdc2 on Tyr-15 in a minimal system devoid of eukaryotic cell components, but does not dephosphorylate other tyrosine-phosphorylated proteins at appreciable rates. In addition, mutations in the putative catalytic site abolish the in vivo activity of cdc25 and its phosphatase activity in vitro. Therefore, cdc25 is a specific protein phosphatase that dephosphorylates tyrosine and possibly threonine residues on p34cdc2 and regulates MPF activation.     

Phosphorylated LIM Kinases Colocalize with Gamma-Tubulin in Centrosomes During Early Stages of Mitosis

LIM kinases (LIMK1 and LIMK2) are LIM domain containing serine/threonine kinases that modulate reorganization of actin cytoskeleton through inactivating phosphorylation of cofilin. The Rho family of small GTPases regulates the catalytic activity of LIMK1 and LIMK2 through activating phosphorylation by ROCK or by p21 kinase. Recent studies have suggested that LIMK1 could play a role in modulation of cellular growth by alteration of the cell cycle in breast and prostate tumor cells; however, the direct mitogenic effects of LIMK1 in these tumor cells is yet to be elucidated. Via immunofluorescence, in this study, we show that phosphorylated LIM kinases (pLIMK1/2) are colocalized with γ-tubulin in the centrosomes during the early mitotic phases of human breast and prostate cancer cells (MDA-MB-231 and DU145); apparent colocalization begins in the centrosomes in prophase. As shown by both bright field (MDA-MB-231) and fluorescent immunohistochemistry (MDA-MB-231 and DU145), pLIMK1/2 does not localize to centrosomes during interphase. By bright field immunohistochemistry, the largest area of the centrosome that is stained with pLIMK1/2 occurs at anaphase. In early telophase, reduced staining of pLIMK1/2 at the spindle poles and concomitant accumulation of pLIMK1/2 at the cleavage furrow begins to occur. In late telophase, loss of staining of pLIMK1/2 and of colocalization with γ-tubulin occurs at the poles and pLIMK1/2 became further concentrated at the junction between the two daughter cells. Co-immunoprecipitation studies indicated that γ-tubulin associates with phosphorylated LIMK1 and LIMK2 but not with dephosphorylated LIMK1 or LIMK2. The results suggest that activated LIMK1/2 may associate with γ-tubulin and play a role in mitotic spindle assembly.     

CDC25B Phosphorylated by pEg3 Localizes to the Centrosome and the Spindle Poles at Mitosis

The phosphatase CDC25B is one of the key regulators that control entry into mitosis throughthe dephosphorylation and subsequent activation of the cyclin-dependent kinases. Here westudy the phosphorylation of CDC25B at mitosis by the kinase pEg3, a member of theKIN1/PAR-1/MARK family. Using mass spectrometry analysis we demonstrate thatCDC25B is phosphorylated in vitro by pEg3 on serine 169, a residue that lies within the Bdomain. Moreover, using phosphoepitope-specific antibodies we show that serine 169 isphosphorylated in vivo, that this phosphorylated form of CDC25B accumulates duringmitosis, and is localized to the centrosomes. This labelling is abrogated when pEg3expression is repressed by RNA interference. Taken together, these results support a model inwhich pEg3 contributes to the control of progression through mitosis by phosphorylation ofthe CDC25 phosphatases.     

The retinoblastoma protein is phosphorylated on multiple sites by human cdc2.

The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.     

Staurosporine is a potent inhibitor of p34cdc2 and p34cdc2-like kinases.

We previously demonstrated that nontransformed cells arrest in the G1 phase of the cell cycle when treated with low concentrations (21 nM) of staurosporine (1). Both normal and transformed cells are blocked in the G2 phase of the cell cycle when treated with higher concentrations (160 nM) of staurosporine (1,2). In the present study, we show that staurosporine inhibits the activity of fractionated p34cdc2 and p34cdc2-like kinases with IC50 values of 4-5 nM. We propose that the G2 phase arrest in the cell cycle caused by staurosporine is due, at least in part, to the inhibition of the p34cdc2 kinases.     

Synthesis of DNA during Mitosis

Growing roots of Zea mays in tritiated thymidine for brief periodsconfirms that G₁ may be eliminated from the mitotic cycle andDNA synthesis may be advanced into telophase but no further,in the fastest dividing cells of the cap initials, but not inthe stele.     

Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase

Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti- PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.     

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.     

cdc2 phosphorylation is required for its interaction with cyclin.

Activation of the cdc2 protein kinase at different stages of the cell cycle is regulated by post-translational modifications and interactions with cyclins. We show that in vitro translated human cdc2 binds very poorly to A and B cyclins, unless it has been preincubated with a Xenopus egg extract. This results in the phosphorylation of cdc2 which allows binding to cyclins. The replacement of Thr161, a residue conserved and phosphorylated in other protein kinases, with valine inhibits cdc2 association with A and B cyclins. In addition, mutations in the amino-terminus of cdc2 and within the conserved 'PSTAIR' region strongly inhibit binding. The Thr161Val mutation causes a lethal phenotype in the fission yeast Schizosaccharomyces pombe, while replacement of Thr161 with glutamic acid, potentially mimicking phosphorylation, causes uncoordination of mitosis and multiple cytokinesis. These results suggest that a threonine phosphorylation/dephosphorylation cycle is involved in regulating cdc2 function.     

The Xenopus XMAP215 and Its Human Homologue TOG Proteins Interact with Cyclin B1 to Target p34cdc2 to Microtubules during Mitosis

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2–cyclinB1 complex). It has previously been demonstrated that the p34cdc2–cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215–cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.     

Phosphorylation of numatrin and other nuclear proteins by cdc2 containing CTD kinase cdc2/p58

Numatrin is a nuclear matrix phosphoprotein whose synthesis and abundance were shown to be regulated during the cell cycle in mitogen-stimulated lymphocytes (Feuerstein, N., and Mond, J. (1987) J. Biol. Chem. 262, 11389-11397). We examined the effect of (a) CTD-kinase, which contains the cdc2 catalytic component (p34) in a complex with a p58 subunit (cdc2/p58) and (b) the M phase-specific histone H1 kinase, which contains the cdc2 kinase in association with a p62 subunit (cdc2/p62), on phosphorylation of numatrin. We show that both cdc2 kinase complexes can phosphorylate numatrin. However, cdc2/p58 at conditions that caused a similar effect to cdc2/p62 on phosphorylation of histone H1 (dpm/micrograms of substrate/micrograms of enzyme) was found to have a 5-25-fold higher catalytic activity in the phosphorylation of numatrin. Analysis of the tryptic phosphopeptide map of numatrin phosphorylated by these cdc2 kinase complexes showed that both kinase complexes phosphorylated two major identical peptides, but minor additional peptides were differentially phosphorylated by each of these kinases. This indicates that under certain experimental conditions cdc2/p58 and cdc2/p62 may express some differences in their catalytic activity. In vitro phosphorylation by CTD kinase of a whole nuclear protein extract from murine fibroblasts showed that numatrin is the most prominent substrate for CTD kinase in this nuclear extract. CTD kinase cdc2/p58 was found to induce significantly the phosphorylation of five other discrete nuclear substrates. Particularly, two nuclear proteins at 75 kDa/pI approximately 6.5 and 85 kDa/pI approximately 5.3, which were not Coomassie Blue stainable, were found to be markedly phosphorylated by CTD kinase. The results of this study call for further study of the role of CTD kinase cdc2/p58 in the phosphorylation of numatrin under physiological conditions and to further characterization of the other nuclear substrates for CTD kinase.     

Beacon interacts with cdc2/cdc28-like kinases

Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism.     

Phosphorylated alpha-synuclein is ubiquitinated in alpha-synucleinopathy lesions

alpha-Synuclein is one of the major components of intracellular fibrillary aggregates in the brains of a subset of neurodegenerative disorders, including Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and Hallervorden-Spatz disease, which are referred to as alpha-synucleinopathies. We have shown previously (Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., Goldberg, M. S., Shen, J., Takio, K., and Iwatsubo, T. (2002) Nat. Cell Biol. 4, 160-164) that alpha-synuclein deposited in synucleinopathy brains is extensively phosphorylated at Ser-129 and migrates at 15 kDa. Here we examined the biochemical characteristics of the additional, higher molecular mass species of phosphorylated alpha-synuclein-positive polypeptides that also are recovered in the Sarkosyl-insoluble fraction of synucleinopathy and migrate at about 22 and 29 kDa. These 22 and 29 kDa bands were positive for three different anti-ubiquitin antibodies and comigrated perfectly with in vitro ubiquitinated alpha-synuclein that may correspond to mono- and diubiquitinated alpha-synuclein, respectively. Furthermore, cyanogen bromide cleavage of the 22 and 29 kDa polypeptides shifted the mobility to 19 and 26 kDa, respectively, and they retained immunoreactivity for both ubiquitin and alpha-synuclein. Finally, protein sequence analysis showed that the 19 kDa band contained two amino-terminal sequences of alpha-synuclein and ubiquitin. These results strongly suggest that phosphorylated alpha-synuclein is targeted to mono- and diubiquitination in synucleinopathy brains, which may have implications for mechanisms of these diseases.