The mitotic spindle is essential for the maintenance of genetic stability, and in budding yeast its assembly and function depend on the Mps1 protein kinase. Mps1p is required for centrosome duplication and the spindle checkpoint. Several recent reports demonstrate that vertebrate Mps1 proteins regulate the spindle checkpoint, but reports conflict regarding their role in centrosome duplication. Here we provide multiple lines of evidence that the human Mps1 protein (hMps1) is required for centrosome duplication. A recently described rabbit polyclonal antibody against hMps1 specifically recognizes centrosomes in a variety of human cell types. Overexpression of a dominant-negative version of hMps1 (hMps1KD) can prevent centrosome duplication in a variety of cell types, and active hMps1 accelerates centrosome reduplication in U2OS cells. Finally, we demonstrate that disruption of hMps1 function with pools of hMps1-specific small interfering RNAs causes a pleiotropic phenotype resulting from the combination of severe mitotic abnormalities and failures in centrosome duplication. This approach demonstrates that hMps1 is required for centrosome duplication and for the normal progression of mitosis, and suggests that the threshold level of hMps1 function required for centrosome duplication is lower than that required for hMps1 mitotic functions.

. In S. cerevisiae, a putative protein-tyrosine phosphatase encoded by PTP2 (22-24) negatively regulates the osmotic stress response pathway, and indirect evidence suggests this occurs by dephosphorylation of Hog1-phosphotyrosine (Hog1-Tyr(P)) (25).We sought to examine further the regulation of MAPK pathways by identifying and characterizing protein phosphatases that act on the HOG pathway in S. cerevisiae. This pathway allows yeast to grow in high osmolarity environments by inducing the expression of osmoprotectants via activation of the MAPK module, Pbs2-Hog1 (Fig. 1) (26). Upstream of the MAPK module is a negative regulator, the "two-component system," comprised of three sequentially acting kinases including Sln1, a plasma membrane bound histidine/aspartyl kinase, Ypd1, a histidine kinase, and Ssk1, an aspartyl kinase (25,27,28). These kinases negatively regulate two MEKKs called Ssk2 and Ssk22 (29). There is also a positive regulator upstream of the MAPK module called Sho1 which activates Pbs2 directly (29). The model for activation of this pathway is as follows. Osmotic

The yeast high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway has been characterized as being activated solely by osmotic stress. In this work, we show that the Hog1 MAPK is also activated by heat stress and that Sho1, previously identified as a membrane-bound osmosensor, is required for heat stress activation of Hog1. The two-component signaling protein, Sln1, the second osmosensor in the HOG pathway, was not involved in heat stress activation of Hog1, suggesting that the Sho1 and Sln1 sensors discriminate between stresses. The possible function of Hog1 activation during heat stress was examined, and it was found that the hog1⌬ strain does not recover as rapidly from heat stress as well as the wild type. It was also found that protein tyrosine phosphatases (PTPs) Ptp2 and Ptp3, which inactivate Hog1, have two functions during heat stress. First, they are essential for survival at elevated temperatures, preventing lethality due to Hog1 hyperactivation. Second, they block inappropriate cross talk between the HOG and the cell wall integrity MAPK pathways, suggesting that PTPs are important for maintaining specificity in MAPK signaling pathways.

The Mps1 protein kinase is required for proper assembly of the mitotic spindle, checkpoint signaling, and several other aspects of cell growth and differentiation. Mps1 regulation is mediated by cell cycle-dependent changes in transcription and protein level. There is also a strong correlation between hyperphosphorylated mitotic forms of Mps1 and increased kinase activity. We investigated the role that autophosphorylation plays in regulating human Mps1 (hMps1) protein kinase activity. Here we report that hyperphosphorylated hMps1 forms are not the only active forms of the kinase. However, autophosphorylation of hMps1 within the activation loop is required for full activity in vitro. The mono-polar spindle-1 (MPS1) gene was identified in Saccharomyces cerevisiae in a screen for mitotic spindle defective mutants (1) and was subsequently shown to encode an essential dual specificity, autophosphorylating protein kinase (2, 3). MPS1 is conserved (4, 5) and is required for a variety of functions during cell growth. The mitotic checkpoint function of Mps1 is conserved among several organisms including yeast, Xenopus laevis, Zebrafish, and humans (6 -13). Recent evidence also suggests that human Mps1 (hMps1) 4 is involved in a DNA damage checkpoint, functioning upstream of Chk2 (14). In S. cerevisiae, duplication of spindle pole bodies requires MPS1 at multiple steps (reviewed in Ref. 15). Similarly, centrosome duplication in mice and humans has been shown to require Mps1 (5, 16), but there is conflicting data on this point (9, 10). Roles for Mps1 in development and in the response to stress have been demonstrated in yeast, Drosophila, and Zebrafish (11,(17)(18)(19)(20)(21).Highly controlled regulation of Mps1 kinase activity is essential for growth. For example, overexpression of MPS1 in S. cerevisiae results in inappropriate checkpoint activation (12, 22), whereas too little Mps1 activity is lethal (2). Mps1 is regulated at both the transcription level, in response to cell cycle progression and cell differentiation (3,4,11,23), and by changes in protein stability (5, 24, 25). The activity of hMps1 rises to an extent greater than what can be explained by the increase in protein level alone during the G 2 /M transition (9, 23). Furthermore, checkpoint activation with nocodazole treatment of cells results in a ϳ30-fold increase in hMps1 activity, whereas the protein level remains similar to untreated mitotic cells (9). Increased hMps1 activity is correlated with more slowly electrophoretically migrating forms thought to be the result of phosphorylation (7, 9). These observations suggest that Mps1 is also regulated by changes in phosphorylation state.Many kinases are activated when phosphorylated within the activation loop (reviewed in Refs. 26 -28). In some cases, this can be catalyzed by autophosphorylation. For example, ERK8 autophosphorylates in vitro on both Thr and Tyr residues for activation, and this is also the likely method for activation in vivo (29). Although it is not clear which other kinases or regulatory s...

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity and protein tyrosine phosphatases (PTPs) in yeasts. In Saccharomyces cerevisiae, two PTPs, Ptp2 and Ptp3, inactivate the MAPKs, Hog1 and Fus3, with different specificities. To further examine the functions and substrate specificities of Ptp2 and Ptp3, we tested whether they could inactivate a third MAPK, Mpk1, in the cell wall integrity pathway. In vivo and in vitro evidence indicates that both PTPs inactivate Mpk1, but Ptp2 is the more effective negative regulator. Multicopy expression of PTP2, but not PTP3, suppressed growth defects due to the MEK kinase mutation, BCK1-20, and the MEK mutation, MKK1-386, that hyperactivate this pathway. In addition, deletion of PTP2, but not PTP3, exacerbated growth defects due to MKK1-386. Other evidence supported a role for Ptp3 in this pathway. Expression of MKK1-386 was lethal in the ptp2⌬ ptp3⌬ strain but not in either single PTP deletion strain. In addition, the ptp2⌬ ptp3⌬ strain showed higher levels of heat stress-induced Mpk1-phosphotyrosine than the wild-type strain or strains lacking either PTP. The PTPs also showed differences in vitro. Ptp2 was more efficient than Ptp3 at binding and dephosphorylating Mpk1. Another factor that may contribute to the greater effectiveness of Ptp2 is its subcellular localization. Ptp2 is predominantly nuclear whereas Ptp3 is cytoplasmic, suggesting that active Mpk1 is present in the nucleus. Last, PTP2 but not PTP3 transcript increased in response to heat shock in a Mpk1-dependent manner, suggesting that Ptp2 acts in a negative feedback loop to inactivate Mpk1.