To cope with life‐threatening high osmolarity, yeast activates the high‐osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 MAP kinase cascade. Activated Hog1 regulates the cell cycle, protein translation, and gene expression. Upstream of the HOG pathway are functionally redundant SLN1 and SHO1 signaling branches. However, neither the osmosensor nor the signal generator of the SHO1 branch has been clearly defined. Here, we show that the mucin‐like transmembrane proteins Hkr1 and Msb2 are the potential osmosensors for the SHO1 branch. Hyperactive forms of Hkr1 and Msb2 can activate the HOG pathway only in the presence of Sho1, whereas a hyperactive Sho1 mutant activates the HOG pathway in the absence of both Hkr1 and Msb2, indicating that Hkr1 and Msb2 are the most upstream elements known so far in the SHO1 branch. Hkr1 and Msb2 individually form a complex with Sho1, and, upon high external osmolarity stress, appear to induce Sho1 to generate an intracellular signal. Furthermore, Msb2, but not Hkr1, can also generate an intracellular signal in a Sho1‐independent manner.

The yeast high osmolarity glycerol (HOG) signaling pathway can be activated by either of the two upstream pathways, termed the SHO1 and SLN1 branches. When stimulated by high osmolarity, the SHO1 branch activates an MAP kinase module composed of the Ste11 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK. To investigate how osmostress activates this MAPK module, we isolated both gain-of-function and loss-of-function alleles in four key genes involved in the SHO1 branch, namely SHO1, CDC42, STE50, and STE11. These mutants were characterized using an HOG-dependent reporter gene, 8xCRE-lacZ. We found that Cdc42, in addition to binding and activating the PAK-like kinases Ste20 and Cla4, binds to the Ste11-Ste50 complex to bring activated Ste20/Cla4 to their substrate Ste11. Activated Ste11 and its HOG pathway-specific substrate, Pbs2, are brought together by Sho1; the Ste11-Ste50 complex binds to the cytoplasmic domain of Sho1, to which Pbs2 also binds. Thus, Cdc42, Ste50, and Sho1 act as adaptor proteins that control the flow of the osmostress signal from Ste20/Cla4 to Ste11, then to Pbs2.

The budding yeast Saccharomyces cerevisiae has at least five signal pathways containing a MAP kinase (MAPK) cascade. The high osmolarity glycerol (HOG) MAPK pathway is essential for yeast survival in high osmolarity environment. This mini-review surveys recent developments in regulation of the HOG pathway with specific emphasis on the roles of protein phosphatases and protein subcellular localization. The Hog1 MAPK in the HOG pathway is negatively regulated jointly by the protein tyrosine phosphatases Ptp2/Ptp3 and the type 2 protein phosphatases Ptc1/Ptc2/Ptc3. Specificities of these phosphatases are determined by docking interactions as well as their cellular localizations. The subcellular localizations of the osmosensors (Sln1 and Sho1), kinases (Pbs2, Hog1), and phosphatases in the HOG pathway are intricately regulated to achieve their specific functions.

Transforming growth factor‐β (TGF‐β), when bound to its specific receptor, activates the transcription factor Smad by phosphorylation. TGF‐β also activates the p38 MAPK pathway, but there seem to be disparate mechanisms for the early p38 activation and delayed p38 activation. In this report, we demonstrate that Smad‐dependent expression of GADD45β is responsible for the delayed activation of p38 by TGF‐β. The GADD45β protein binds and activates MTK1 (= MEKK4), which is a member of the MAPKKK family kinases and an upstream activator of the p38 MAPK cascade. Both TGF‐β‐induced GADD45β expression and the delayed p38 activation require functional Smad proteins. Antisense inhibition of GADD45β expression suppresses the TGF‐β‐induced delayed p38 activation, whereas overexpression of GADD45β activates the p38 MAPK via MTK1. Expression of the angiogenesis inhibitor thrombospondin‐1 (TSP‐1) is induced by TGF‐β via Smad‐dependent p38 activation. Thus TGF‐β‐induced p38 activation, mediated by GADD45β expression, may play an important role in the biological effects of TGF‐β.

Hsk1, Saccharomyces cerevisiae Cdc7-related kinase in Shizosaccharomyces pombe, is required for G1/S transition and its kinase activity is controlled by the regulatory subunit Dfp1/Him1. Analyses of a newly isolated temperature-sensitive mutant, hsk1-89, reveal that Hsk1 plays crucial roles in DNA replication checkpoint signaling and maintenance of proper chromatin structures during mitotic S phase through regulating the functions of Rad3 (ATM)-Cds1 and Rad21 (cohesin), respectively, in addition to expected essential roles for initiation of mitotic DNA replication through phosphorylating Cdc19 (Mcm2). Checkpoint defect in hsk1-89 is indicated by accumulation of cut cells at 30°C. hsk1-89 displays synthetic lethality in combination with rad3 deletion, indicating that survival of hsk1-89 depends on Rad3-dependent checkpoint pathway. Cds1 kinase activation, which normally occurs in response to early S phase arrest by nucleotide deprivation, is largely impaired in hsk1-89. Furthermore, Cds1-dependent hyperphosphorylation of Dfp1 in response to hydroxyurea arrest is eliminated in hsk1-89, suggesting that sufficient activation of Hsk1-Dfp1 kinase is required for S phase entry and replication checkpoint signaling. hsk1-89 displays apparent defect in mitosis at 37°C leading to accumulation of cells with near 2C DNA content and with aberrant nuclear structures. These phenotypes are similar to those of rad21-K1 and are significantly enhanced in a hsk1-89 rad21-K1 double mutant. Consistent with essential roles of Rad21 as a component for the cohesin complex, sister chromatid cohesion is partially impaired in hsk1-89, suggesting a possibility that infrequent origin firing of the mutant may affect the cohesin functions during S phase. INTRODUCTIONDNA replication needs to be stringently regulated for cell growth and cell division to occur in a coordinated manner (Stillman, 1996). Initiation of DNA replication requires assembly of multiprotein complexes at chromosomal replication origins during late M to early G1 phase Newlon, 1997). This complex, termed prereplicative complex (preRC), includes ORC (Bell and Stillman, 1992), Cdc6 (Cocker et al., 1996), and MCM proteins (Tye, 1994;Kearsey et al., 1995;Chong et al., 1996;Donovan et al., 1997), which are conserved from yeasts to human. After cells pass Start, the preRC is activated and DNA synthesis is initiated at replication origins. This process is accompanied with dissociation of Cdc6 (Cocker et al., 1996;Tanaka et al., 1997) and of at least some components of the MCM complex from origins Aparicio et al., 1997;Kubota et al., 1997;Tanaka et al., 1997), resulting in a postreplicative complex (postRC), which is inactive until the next cell cycle. The firing of origins requires actions of at least two distinct serine/threonine kinases, namely, G1/S-specific CDK-Cyclin (Nasmyth, 1996;Stillman, 1996) and Cdc7-Dbf4 (Hartwell, 1971, 1973.Saccharomyces cerevisiae CDC7 encodes a serine/threonine protein kinase required for the onset of DNA replication (Hollingsworth et al., 1992;Jackso...