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Melanoma is highly heterogeneous with diverse genomic alterations and partial therapeutic responses. Emergence of drug-resistant tumor cell clones accompanied with high AXL expression level is one of the major challenges for anti-tumor clinical care. Recent studies have demonstrated that high AXL expression in melanoma cells mediated drug-resistance, epithelial-mesenchymal transition (EMT) and elevated survival of cancer stem cells (CSCs). Given that we have identified several non-steroidal anti-inflammatory drugs (NSAIDs) including Aspirin potently induce the degradation of AXL, we questioned whether NSAIDs could counteract the AXL-mediated neoplastic phenotypes. Here we found NSAIDs downregulate PKA activity via the PGE2 /EP2/cAMP/PKA signaling pathway and interrupt the PKA-dependent interaction between CDC37 and HSP90, resulting in an incorrect AXL protein folding and finally AXL degradation through the ubiquitination-proteasome system (UPS) pathway. Furthermore, NSAIDs not only sensitized the MEK inhibitor treatment, but also reduced EMT and relapse mediate by AXL in tumor tissue. Our findings suggest that the combination of inhibitors and NSAIDs, especially Aspirin, could be a simple but efficient modality to treat melanoma in which AXL is a key factor for drug-resistance, metastasis, and relapse.

Homeodomain-interacting protein kinase 2 (HIPK2) is a highly conserved, constitutively active Ser/Thr protein kinase that is involved in various important biological processes. HIPK2 activates itself by auto-phosphorylation during its synthesis, and its activity is mainly controlled through modulation of its expression by ubiquitin-dependent degradation. By comparing the expression of wild-type and kinase-defective HIPK2, we have recently described a novel mechanism of HIPK2 regulation that is based on preferential co-translational degradation of kinase-defective versus wild-type HIPK2. Here, we have addressed this novel regulatory mechanism in more detail by focusing on the possible involvement of chaperones. Our work shows that HIPK2 is a client of the CDC37-HSP90 chaperone complex and points to a novel role of CDC37 in the co-translational degradation of a client protein.

Heat shock protein 90 (HSP90) is an indispensable molecular chaperone that facilitates the maturation of numerous oncoproteins in cancer cells, including protein kinases, ribonucleoproteins, steroid hormone receptors, and transcription factors. Although over 30 HSP90 inhibitors have steadily entered clinical trials, further clinical advancement has been restricted by their limited efficacy, inevitable heat shock response, and multiple side-effects, likely induced via an ATP inhibition mechanism. Since both ATP and various co-chaperones play essential roles in the HSP90 chaperone cycle to achieve integrated function, optimal therapeutics require an understanding of the dynamic interactions among HSP90, ATP, and cochaperones. To date, continuous research has promoted the exploration of the cochaperone cell division cycle 37 (CDC37) as a kinase-specific recognizer and has shown that the HSP90-CDC37-kinase complex is particularly relevant in cancers. Indeed, disrupting the HSP90-CDC37-kinase complex, rather than totally blocking the ATP function of HSP90, is emerging as an alternative way to avoid the limitations of current inhibitors. In this review, we first briefly introduce the HSP90-CDC37-kinase cycle and present the currently available approaches for inhibitor development targeting this cycle and provide insights into selective regulation of the kinase clients of HSP90 by more directional ways.

Myoglobin (Mb) maturation involves heme incorporation as a final step. We investigated a role for heat shock protein (hsp) 90 in Mb maturation in C2C12 skeletal muscle myoblasts and cell lines. We found the following: 1) Hsp90 directly interacts preferentially with heme-free Mb both in purified form and in cells. 2) Hsp90 drives heme insertion into apoprotein-Mb in an ATP-dependent process. 3) During differentiation of C2C12 myoblasts into myotubes, the apo-Mb-hsp90 complex associates with 5 cell cochaperons, Hsp70, activator of hsp90 ATPase protein 1 (Aha1), alanyl-tRNA synthetase domain containing 1 (Aarsd1), cell division cycle 37 (Cdc37), and stress induced phosphoprotein 1 (STIP1) in a pattern that is consistent with their enabling Mb maturation. 4) Mb heme insertion was significantly increased in cells that had a functional soluble guanylyl cyclase (sGC)-cGMP signaling pathway and was diminished upon small interfering RNA knockdown of sGCβ1 or upon overexpression of a phosphodiesterase to prevent cGMP buildup. Together, our findings suggest that hsp90 works in concert with cochaperons (Hsp70, Aha1, Aarsd1, STIP1, and Cdc37) and an active sGC-cGMP signaling pathway to promote heme insertion into immature apo-Mb, and thus generate functional Mb during muscle myotube formation. This fills gaps in our understanding and suggests new ways to potentially control these processes.—Ghosh, A., Dai, Y., Biswas, P., Stuehr, D. J. Myoglobin maturation is driven by the hsp90 chaperone machinery and by soluble guanylyl cyclase.

Cell division cycle 37 (Cdc37) is an important partner for heat shock protein 90 (HSP90), assisting in molecular chaperone activities, particularly with regard to the regulation of protein kinases. Given its influence on cell growth pathways, Cdc37 has been discussed as a potential intermediate in carcinogenesis. However, to date, the potential functional roles and molecular mechanisms by which Cdc37 regulates cell survival in colorectal carcinoma (CRC) remain unclear. Here, we investigated the expression of Cdc37 and its clinical significance in CRC, and systematically explored the role and the underlying mechanism of Cdc37 in CRC cell survival both in vitro and in vivo. Our results showed that Cdc37 was remarkably up-regulated in CRC, which facilitated cell survival mainly by promoting cell proliferation, G1-S transition, and inhibiting cell apoptosis. Our data further indicated that Cdc37 increased the stability of cyclin-dependent kinase 4 (CDK4) to activate the retinoblastoma 1 (RB1) signaling pathway, followed by increased expression of Bcl-2 and Bcl-xL, which ultimately promoted cell survival in CRC. Moreover, knockdown of CDK4 reversed the Cdc37-mediated effect in promoting the progression of CRC. Our findings showed that Cdc37 played a critical role in promoting CRC cell survival by increasing CDK4 stability to activate the RB1 signaling pathway. Thereby, Cdc37 might serve as a potential therapeutic target in CRC patients.

By catalysing reversible phosphorylation of their substrates, protein kinases play pleiotropic roles in cells and act as predominant arbiters in the coordination of cellular responses to their environment. Despite large variation in biological functions between kinases, there is great structural conservation across the human kinome. Besides structural aspects, substrate specificity relies on dynamic factors such as concentration, activity and localisation. This article discusses these fundamental aspects of protein kinases as related to cellular signalling while also taking note of the less commonly discussed members of the class such as pseudokinases and ectokinases. The complexity of signalling networks is demonstrated by a discussion on the crosstalk involved between the metabolic and signal transduction pathways. By having roles in such processes as cell growth, proliferation and death, protein kinases are at the centre of the regulation of the cell and therefore also at the heart of many human diseases when dysregulated. Key Concepts Protein kinases are enzymes that catalyse the phosphorylation of a protein substrate. Reversible phosphorylation is a key feature of cellular signalling. There are some highly conserved structural motifs across the human kinome despite great differences in biological functions. Activation of a kinase is dependent upon assembly of the regulatory spine. Structural and dynamic factors contribute to substrate selectivity. Kinases are present both within the cell and in extracellular spaces (ectokinases). Crosstalk with other post-translational modifications and between other pathways makes protein kinases participants in nearly every physiological process. Keywords: protein kinase; pseudokinase; signal transduction; kinome; metabolism; phosphorylation; post-translational modification

Flow cytometry, in conjunction with immunoprecipitation (IP-FCM), is suggested to have some advantages to conventional IP-western blot technology in analyzing protein complexes. In this paper, to further examine its practicability, we test the use of IP-FCM in detecting the HSP90 complex, which has gained importance in drug research and development and involves more than a dozen components. We found that IP-FCM could effectively detect HSP70, p23, Cdc37, and Cdk6 components in the HSP90 complex naturally formed in U937 cells when this complex was captured by anti-HSP90 antibody-coated polystyrene microspheres. IP-FCM could also detect alteration in components caused by treating cells with HSP90 inhibitors. In a cell-free environment, IP-FCM could detect the direct effects of ATP and/or HSP90 inhibitors (17-N-allylamino-17-demethoxygeldanamycin or celastrol) in causing component dissociation and the time- and dose-effects of inhibitor-caused dissociation. IP-FCM is a practical and powerful platform for analyzing HSP90 complex components, and is thus a useful tool in studying HSP90 complex function and screening inhibitors. © 2013 International Society for Advancement of Cytometry

It is hypothesized that autophagy, a global catabolic pathway which is highly conserved from yeast to man, plays an important role in many bioprocesses. Though autophagy is known to be induced by either nutrient starvation or treatment with the drug rapamycin, it is not clear whether the two modes of induction have the same long-term impact in the cell, particularly in the biotechnologically important filamentous fungi. Here, we compare the overall proteomes from the carbon-starved (G−) and rapamycin treated (R+) model fungus Aspergillus nidulans. From about 1,100 visualized protein spots, we conservatively selected a total of 26 proteins with significant different expression. To highlight, increased levels of glucosidases and decreased levels of N-acetylglucosamine pyrophosphorylase were observed, suggesting degradation of the fungal cell wall as an alternate carbon source for both modes of induction. Cdc37 was reduced in expression while 14-3-3 ArtA was increased, implying regulation of polar growth, while also potentially regulating autophagy negatively via PKA or Tor. Other proteins included aspartate transaminase, tryptophan synthase B (TrpB), glycylpeptide N-tetradecanoyltransferase (Nmt1), and aldehyde dehydrogenase (aldA). More interestingly, the majority of the identified proteins (16 of 26) were uniquely expressed in elevated levels in G−. A novel predicted protein from AN8223 which has no sequence homology to other organisms is also implicated to be involved in carbon-starvation. Thus, proteomic data here show that in A. nidulans, rapamycin-induced autophagy and carbon-starvation induced autophagy share some effectors for cell survival, but predominantly involve different long-term effectors. Biotechnol. Bioeng. 2011;108: 2705–2715. © 2011 Wiley Periodicals, Inc.

Race-specific disease resistance in plants depends on the presence of resistance (R) genes. Most R genes encode NB-ARC-LRR proteins that carry a C-terminal leucine-rich repeat (LRR). Of the few proteins found to interact with the LRR domain, most have proposed (co)chaperone activity. Here, we report the identification of RSI2 (Required for Stability of I-2) as a protein that interacts with the LRR domain of the tomato R protein I-2. RSI2 belongs to the family of small heat shock proteins (sHSPs or HSP20s). HSP20s are ATP-independent chaperones that form oligomeric complexes with client proteins to prevent unfolding and subsequent aggregation. Silencing of RSI2-related HSP20s in Nicotiana benthamiana compromised the hypersensitive response that is normally induced by auto-active variants of I-2 and Mi-1, a second tomato R protein. As many HSP20s have chaperone properties, the involvement of RSI2 and other R protein (co)chaperones in I-2 and Mi-1 protein stability was examined. RSI2 silencing compromised the accumulation of full-length I-2 in planta, but did not affect Mi-1 levels. Silencing of heat shock protein 90 (HSP90) and SGT1 led to an almost complete loss of full-length I-2 accumulation and a reduction in Mi-1 protein levels. In contrast to SGT1 and HSP90, RSI2 silencing led to accumulation of I-2 breakdown products. This difference suggests that RSI2 and HSP90/SGT1 chaperone the I-2 protein using different molecular mechanisms. We conclude that I-2 protein function requires RSI2, either through direct interaction with, and stabilization of I-2 protein or by affecting signalling components involved in initiation of the hypersensitive response.

Protein kinases play important roles in almost all major gnaling and regulatory pathways of eukaryotic organisms. Members in the family of protein kinases make up a substantial fraction of eukaryotic proteome. Analysis of the protein kinase repertoire (kinome) would help in the better understanding of the regulatory processes. In this rticle,we report the identification and analysis of the repertoire of protein kinases in the intracellular parasite Entamoeba histolytica. Using a combination of various sensitive sequence search methods and manual analysis, we have identified a set of 307 protein kinases in E. histolytica genome. We have classified these protein kinases into different subfamilies originally defined by Hanks and Hunter and studied these kinases further in the context of noncatalytic domains that are tethered to catalytic kinase domain. Compared to other eukaryotic organisms, protein kinases from E. histolytica vary in terms of their domain organization and displays features that may have a bearing in the unusual biology of this organism. Some of the parasitic kinases show high sequence similarity in the catalytic domain region with calmodulin/calcium dependent protein kinase subfamily. However, they are unlikely to act like typical calcium/calmodulin dependent kinases as they lack noncatalytic domains characteristic of such kinases in other organisms. Such kinases form the largest subfamily of kinases in E. histolytica. Interestingly, a PKA/PKG-like subfamily member is tethered to pleckstrin homology domain. Although potential cyclins and cyclindependent kinases could be identified in the genome the likely absence of other cell cycle proteins suggests unusual nature of cell cycle in E. histolytica. Some of the unusual features recognized in our analysis include the absence of MEK as a part of the Mitogen Activated Kinase signaling pathway and identification of transmembrane region containing Src kinase-like kinases. Sequences which could not be classified into known subfamilies of protein kinases have unusual domain architectures. Many such unclassified protein kinases are tethered to domains which are Cysteine-rich and to domains known to be involved in protein–protein interactions. Our kinome analysis of E. histolytica suggests that the organism possesses a complex protein phosphorylation network that involves many unusual kinases.

The CK2-dependent phosphorylation of Ser13 in cell division cycle protein 37 (Cdc37), a kinase-specific heat shock protein 90 (Hsp90) cochaperone, has previously been reported to be essential for the association of Cdc37 with signaling protein kinases [Bandhakavi S, McCann RO, Hanna DE & Glover CVC (2003) J Biol Chem278, 2829–2836; Shao J, Prince T, Hartson SD & Matts RL (2003) J Biol Chem278, 38117–38220; Miyata Y & Nishida E (2004) Mol Cell Biol24, 4065–4074]. Here we describe a new phospho-specific antibody against Cdc37 that recognizes recombinant purified Cdc37 only when incubated with CK2 in the presence of Mg2+ and ATP. The replacement of Ser13 in Cdc37 by nonphosphorylatable amino acids abolished binding to this antibody. The antibody was specific for phosphorylated Cdc37 and did not crossreact with other CK2 substrates such as Hsp90 and FK506-binding protein 52. Using this antibody, we showed that complexes of Hsp90 with its client signaling kinases, Cdk4, MOK, v-Src, and Raf1, contained the CK2-phosphorylated form of Cdc37 in vivo. Immunofluorescent staining showed that Hsp90 and the phosphorylated form of Cdc37 accumulated in epidermal growth factor-induced membrane ruffles. We further characterized the phosphorylation of Cdc37 using phospho-affinity gel electrophoresis. Our analyses demonstrated that the CK2-dependent phosphorylation of Cdc37 on Ser13 caused a specific gel mobility shift, and that Cdc37 in the complexes between Hsp90 and its client signaling protein kinases was in the phosphorylated form. Our results show the physiological importance of CK2-dependent Cdc37 phosphorylation and the usefulness of phospho-affinity gel electrophoresis in protein phosphorylation analysis.

Simian virus 40 large T antigen is a J-domain-containing protein with multiple functions. Among its numerous activities, T antigen can bind heat shock cognate 70 (hsc70) but the biological significance of this interaction has not been fully understood. Here, we show that T antigen can act as an hsc70 co-chaperone enhancing the protein-folding ability of the hsc70 chaperone machine. We also show that T antigen exerts its function in collaboration with the mammalian homologue of DnaJ. Moreover, we show that the participation of T antigen in the hsc70 chaperone machine has cell-type-specific characteristics.

Cdc37 is a molecular chaperone that interacts with a range of clients and co-chaperones, forming various high molecular mass complexes. Cdc37 sequence homology among species is low. High homology between yeast and metazoan proteins is restricted to the extreme N-terminal region, which is known to bind clients that are predominantly protein kinases. We show that despite the low homology, both Saccharomyces cerevisiae and human Cdc37 are able to substitute for the Schizosaccharomyces pombe protein in a strain deleted for the endogenous cdc37 gene. Expression of a construct consisting of only the N-terminal domain of S. pombe Cdc37, lacking the postulated heat-shock protein (Hsp) 90-binding and homodimerization domains, can also sustain cellular viability, indicating that Cdc37 dimerization and interactions with the cochaperone Hsp90 may not be essential for Cdc37 function in S. pombe. Biochemical investigations showed that a small proportion of total cellular Cdc37 occurs in a high molecular mass complex that also contains Hsp90. These data indicate that the N-terminal domain of Cdc37 carries out essential functions independently of the Hsp90-binding domain and dimerization of the chaperone itself.

This chapter covers two types of regulatory signal transduction elements in Mycobacterium tuberculosis: (i) histidine protein kinase response regulators, referred to as two-component systems, and (ii) eucaryotic-type Ser/Thr protein kinases (STPKs). The chapter is divided into two parts, covering the two types of phosphotransfer signaling systems individually. The prototypical two-component systems consist of a histidine protein kinase (often functioning as an environmental sensor), which, as a manifestation of signal transduction, phosphorylates an aspartate residue on its cognate response regulator (a transcriptional regulator or a regulator of other proteins). The observations that inactivation of five different two-component systems increases the ability of M. tuberculosis to kill the murine host or enhances the growth of the mutant strains in mice or macrophages may reflect evolutionary adaptations of M. tuberculosis important for propagation and infectious cycle of the pathogen. Protein phosphorylation is carried out by specific protein kinases and is coupled to dephosphorylation reactions carried out by protein phosphatases. The eukaryotic protein kinases and phosphatases form the backbone of this signal transduction pathway. M. tuberculosis encodes and expresses at least eight eukaryotic-like protein kinases, and six proteins can be phosphorylated in vitro, suggesting the presence of functional kinases in M. tuberculosis. Interference with the normal immune response of the host during mycobacterial infection could be mediated by disabling host signaling pathways.

p23 is a regulatory co-chaperone of heat shock protein (Hsp) 90, but can also act as a general molecular chaperone by itself. Using novel point mutations of p23 that disrupt its interaction with Hsp90 we found its co-chaperone function to be required for its inhibitory effect on glucocorticoid receptor (GR). The C-terminal region of p23, which is required for its chaperone activity, is dispensable for inhibition of GR. Importantly, similar results were obtained with a constitutively active GR. Thus, the action of p23 on the nuclear stage of GR regulation requires its Hsp90 co-chaperone function, but not its chaperone activity.

We report here the identification of the homologous gene pair ZDS1,2 as multicopy suppressors of a temperature-sensitive allele (cka2-13ts) of the CKA2 gene encoding the α′ catalytic subunit of protein kinase CK2. Overexpression of ZDS1,2 suppressed the temperature sensitivity, geldanamycin (GA) sensitivity, slow growth, and flocculation of multiple cka2 alleles and enhanced CK2 activity in vivo toward a known physiological substrate, Fpr3. Consistent with the existence of a recently described positive feedback loop between CK2 and Cdc37, overexpression of ZDS1,2 also suppressed the temperature sensitivity, abnormal morphology, and GA sensitivity of a CK2 phosphorylation-deficient mutant of CDC37, cdc37-S14A, as well as the GA sensitivity of a cdc37-1 allele. A likely basis for all of these effects is our observation that ZDS1,2 overexpression enhances Cdc37 protein levels. Activation of the positive feedback loop between CK2 and Cdc37 likely contributes to the pleiotropic nature of ZDS1,2, as both CK2 and Cdc37 regulate diverse cellular functions.

Vasopressin regulates the urea channel protein UT-A1 by phosphorylation at Ser84 and Ser486. What kinases are responsible for these phosphorylation events remains an open question. To identify protein kinases that interact with UT-A1 in IMCD, we employed crosslinking with BSOCOES, UT-A1 immunoprecipitation, SDS-PAGE separation, and quantitative mass spectrometry of trypsin digests of gel pieces. The peptides passing stringent spectral quality thresholds were quantified (label-free) to identify those with high signal:noise (UTA1 antibody/preimmune IgG>4). This identified four protein kinases bound to the UT-A1 (Phosphorylase Kinase [PhK], Calmodulin-Dependent Kinase 2δ[CAMK2], CDC42-Binding Protein Kinase β[CDC42BPK] and mTOR). In vitro phosphorylation assays were carried out by incubating these kinases or Protein Kinase A (purified, active recombinant) with synthetic peptides containing the known phosphorylation sites in UT-A1. Phosphorylation was determined by immunoblotting using phospho...

The Wee1 protein kinase negatively regulates entry into mitosis by mediating the inhibitory tyrosine phosphorylation of Cdc2-cyclin B kinase. The stability and activity of Wee1 from the fission yeast Schizosaccharomyces pombe is critically dependent on functional Hsp90 chaperones. Here we identify two related tyrosine protein kinases, Mik1 from fission yeast and its Saccharomyces cerevisiae homolog Swe1, as Hsp90 substrates and show that the kinase domain is sufficient to mediate this interaction. Morphological and biochemical defects arising from overexpression of the kinases in fission yeast are suppressed in the conditional Hsp90 mutant swo1-26. A subset of all three kinases is associated with the Hsp90 cochaperones cyclophilin 40 and p23. Under conditions of impaired chaperone function or treatment with the Hsp90 inhibitory drug geldanamycin, intracellular levels of the kinases are reduced and the proteins become rapidly degraded by the proteasome machinery, indicating that Wee1, Mik1 and Swe1 require Hsp90 heterocomplexes for their stability and maintenance of function.

Basal and stress-induced synthesis of the components of the highly conserved heat shock protein Hsp90 chaperone complex require the heat shock transcription factor (HSF); Saccharomyces cerevisiae cells expressing the HSF allele HSF(1-583) reversibly arrest growth at 37 degrees C in the G(2)/M phase of the cell cycle due to diminished expression of these components. A suppressor mutant capable of restoring high-temperature growth to HSF(1-583) cells was identified, harboring a disruption of the SCH9 protein kinase gene, homologous to the protein kinase A and protein kinase B/Akt families of mammalian growth control kinases. Loss of Sch9 in HSF(1-583) cells derepresses Hsp90 signal transduction functions as demonstrated by restoration of transcriptional activity by the mammalian glucocorticoid receptor and the heme-dependent transcription factor Hap1, and by enhanced pheromone-dependent signaling through the Ste11 mitogen-activated protein kinase (MAPK). Moreover, Sch9-deficient cells with normal levels of Hsp90 chaperone complex components display hyperactivation of the pheromone response MAPK pathway in the absence of pheromone. These results demonstrate that the evolutionarily conserved function of the Hsp90 chaperone complex as a signal transduction facilitator is modulated by a growth regulatory kinase.

The various mitogen-activated protein (MAP) kinases have central roles in the signalling pathways of T lymphocytes. Their activation is uniquely dependent on dual phosphorylation of a serine/threonine and a tyrosine residue and is regulated by several levels of kinases in parallel cascades. In addition, both the MAP kinases and their upstream, activating kinases are regulated by several phosphatases. Although each of the MAP kinases have many cytoplasmic substrates, their ability to translocate to the nucleus means that they can transmit signals from the cytoplasm directly to transcription factors, which are sometimes nuclear bound. The MAP kinase cascades are activated in T lymphocytes by a variety of different external stimuli. They play an important role in transducing both the signal from T cell receptor and costimulatory molecules, on the T cell surface, and are able to regulate several of the transcription factors controlling the expression of critical genes, including that for IL-2. This review examines how the activation of several MAP kinases is regulated, their role in signal transduction initiated by a variety of stimuli, and how this may lead to different cellular responses.

Hsp90 is an abundant and constitutively expressed stress protein and molecular chaperone. Here we dissected human hsp90 into three major domains to identify the putative chaperone site at which hsp90 binds unfolded polypeptide. Surprisingly, both the N-terminal and the C-terminal domain of hsp90 prevent the aggregation of denatured polypeptides. The chaperone activity of the N-domain is inhibited by geldanamycin, a specific inhibitor of hsp90-mediated protein refolding. While both domains suppress protein aggregation, only the C-domain binds an antigenic peptide derived from VSV G. Based on these results, hsp90 may be the first chaperone to contain two independent chaperone sites with differential specificity.

Ste20/PAK serine/threonine protein kinases have been suggested as playing essential roles in cell signalling and morphogenesis as potential targets of Cdc42 and Rac GTPases. We have isolated and characterized the Saccharomyces cerevisiae SKM1 gene, which codes for a novel member of this family of protein kinases. The amino acid sequence analysis of Skm1p revealed the presence of a PH domain and a putative p21-binding domain near its amino terminus, suggesting its involvement in cellular signalling or cytoskeletal functions. However, deletion of SKM1 produced no detectable phenotype under standard laboratory conditions. Moreover, disruption of each of the two other S. cerevisiae Ste20/PAK-like kinase-encoding genes, STE20 and CLA4, in skm1 backgrounds, showed that Skm1p is not redundant with Ste20p or Cla4p. Interestingly, overexpression of SKM1 led to morphological alterations, indicating a possible role for this protein in morphogenetic control. Furthermore, overproduction of Skm1p lacking its N-terminus caused growth arrest. This effect was also seen when similarly truncated versions of Ste20p or Cla4p were overexpressed. We further observed that overproduction of this C-terminal fragment of Skm1p complements the mating defect of a ste20 mutant strain. These results suggest that the N-terminal domains of S. cerevisiae Ste20/ PAK-like protein kinases share a negative regulatory function and play a role in substrate specificity.

Two new kinases of Dictyostelium discoideum were identified by screening of a λgt11 expression library with a phosphotyrosine specific antibody. Amino-acid sequences derived from cDNA and genomic clones indicate that DPYK3 is a protein of 150 kDa and DPYK4, a protein of 75 kDa. The C-terminal fragments of each protein were produced in Escherichia coli and shown to be autocatalytically phosphorylated at tyrosine residues. A common feature of these kinases is the presence of two different sequence stretches in tandem that are related to kinase catalytic domains. The sequence relationships of DPYK3 and 4 to other protein kinases, and the positions of their catalytic domain sequences within the phylogenetic tree of protein kinases were analysed. Domains I of both kinases and domain II of DPYK3 constitute, together with the catalytic domains of two previously described tyrosine kinases of D. discoideum , a branch of their own, separate from the tyrosine kinase domains in sensu strictu. Domain II in DPYK4 is found on a different branch close to serine/threonine kinases.

Protein synthesis is regulated in response to environmental stimuli by covalent modification, primarily phosphorylation, of components of the translational machinery. Phosphorylation of the alpha subunit of eIF-2 is one of the best-characterized mechanisms for down-regulating protein synthesis in higher eukaryotes in response to various stress conditions. Three distinct protein kinases regulate protein synthesis in eukaryotic cells by phosphorylating the alpha subunit of eIF-2 at serine-51. There are two mammalian eIF-2alpha kinases: the double-stranded RNA-dependent kinase (PKR) and heme-regulated inhibitor kinase (HRI), and the yeast GCN2. The regulatory mechanisms and the molecular sizes of these eIF-2alpha kinases are different. The expression of PKR is induced by interferon, and the kinase activity is stimulated by low concentrations of double-stranded RNA. HRI is activated under heme-deficient conditions. Yeast GCN2 is activated by amino acid starvation. The phosphorylation of eIF-2alpha results in the shutdown of protein synthesis. Nevertheless, the eIF-2alpha kinases can regulate both global as well as specific mRNA translation. Inhibition of protein synthesis correlates with eIF-2alpha phosphorylation in response to a wide variety of different stimuli, including heat shock, serum deprivation, glucose starvation, amino acid starvation, exposure to heavy metal ions, and viral infection. Finally, recent studies suggest a role for eIF-2alpha phosphorylation in the control of cell growth and differentiation.

Expression of proteins in eucaryotic systems is often the only way to ensure the correct folding and processing necessary for protein function. Heterologous proteins, however, are commonly retained in the secretory pathway, so that secreted product yield is low despite a high level of transcription. A major limiting step in protein secretion is protein folding in the lumen of the endoplasmic reticulum. This process is assisted by accessory macromolecules resident in this compartment, including chaperones such as the hsp70 homologue binding protein (BiP). Although induction of foreign proteins in yeast initially elicits a transient increase in local chaperone concentration, long-term protein expression lowers both chaperone and secreted product. A mechanistic model that can account for the experimentally observed role of BiP in secretion and the effects of BiP overexpression on the secretory pathway is described here. The model predicts that equimolar synthesis of chaperone and foreign protein should optimize protein secretion.

In the yeast Saccharomyces cerevisiae, the heterotrimeric G protein transduces the mating pheromone signal from a cell-surface receptor. Free G beta gamma then activates a mitogen-activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal-transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone-induced signal which leads cells to hormone-induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone-induced signal transduction, and overexpression of STE50 intensifies the pheromone-induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the alpha-pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two-hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae.

The rat cdc37-related gene product (RCdc37), which is possibly involved in the regulation of cell cycle progression, contains a putative basic/leucine zipper (bZIP) domain in its N-terminal portion. In this study, we have identified a rat genomic sequence which can interact with RCdc37 using an in vitro binding assay. The specificity of this interaction was confirmed by gel retardation experiments. These results raise a possibility that RCdc37 might play an important role in the control of cell cycle progression via a sequence-specific DNA binding mechanism.

We have cloned a new gene, SCP160, from Saccharomyces cerevisiae, the deduced amino acid sequence of which does not exhibit overall similarity to any known yeast protein. A weak resemblance between the C-terminal part of the Scp160 protein and regulatory subunits of cAMP-dependent protein kinases from eukaryotes as well as the pstB protein of Escherichia coli was observed. The SCP160 gene resides on the left arm of chromosome X and codes for a polypeptide of molecular weight around 160 kDa. By immunofluorescence microscopy the Scp160 protein appears to be localized to the nuclear envelope and to the endoplasmic reticulum (ER). However, no signal sequence or membrane-spanning region exists, suggesting that the Scp160 protein is attached to the cytoplasmic surface of the ER-nuclear envelope membranes. Disruption of the SCP160 gene is not lethal but results in cells of decreased viability, abnormal morphology and increased DNA content. This phenotype is not reversible by transformation with a plasmid carrying the wild-type gene. Crosses of SCP160 deletion mutant strains among each other or with unrelated strains lead to irregular segregation of genetic markers. Taken together the data suggest that the Scp160 protein is required during cell division for faithful partitioning of the ER-nuclear envelope membranes which in S. cerevisiae enclose the duplicated chromosomes.

We have isolated a single gene from the yeast Saccharomyces cerevisiae encoding a potential 800 amino acid polypeptide of calculated M(r) 90,098 Da. This protein consists of an N-terminal region that shares significant homology with the catalytic domains of several serine- and threonine-specific protein kinases, as well as a large, unique, C-terminal domain of unknown function. Haploid disruption mutants are viable and do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients or osmotic strength. Due to the apparent structural similarity between this kinase and the protein products of the KIN1 and KIN2 genes, we have chosen to name this new gene KIN3.

The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins.

Summary A novel gene, IRE1, of Saccharomyces cerevisiae was cloned through genetic complementation of a myo- inositol auxotrophic mutant. The predicted amino acid sequence indicated that IRE1 encodes a protein of 126983 Da with two highly hydrophobic regions, probably a signal sequence and a membrane-spanning region. The carboxy-terminal region of IRE1 showed close sequence similarity to the catalytic domains of protein kinases. Disruption of the IRE1 locus caused myo-inositol auxotrophy. The IRE1 product is very likely a protein kinase required for myo-inositol synthesis.

The cell division cycle gene CDC 15 is essential for the late nucler division in the yeast Saccharomyces cerevisiae. The amino acid sequence of the 974 amino acids/110 kDa CDC 15 gene product, as deduced from the nucletide sequence, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns specific for protein serine/theonine kinases besides those for protein tyrosine kinases. Many protein kinases non-essential for growth are known. CDC 15 represents an essential protein kinase like CDC 7 and CDC 28. A carboxyterminal deletion of 32 amino acids renders the protein inactive.

Protein kinases represent a diverse family of enzymes that play a critical role in regulation. Among nearly 100 known protein kinases, the cAMP-dependent enzyme is best understood biochemically. Unlike other protein kinases, cAMP-dependent protein kinase consists of two different types of subunits that dissociate, a regulatory subunit (R), which is the receptor for cAMP, and a catalytic subunit (C). In the absence of cAMP, the enzyme exists as an inactive tetramer, R2C2. The binding of intracellular cAMP to the R subunit decreases the affinity of the R subunit for the C subunit by approximately four orders of magnitude and, under physiological conditions, leads to dissociation of the holoenzyme into R2(cAMP)4 dimer and two free C subunits that are catalytically active. Mutants of the cAMP metabolism, adenylate cyclase and cell cycle mutants, provided further information about protein synthesis and cellular growth in Saccharomyces cerevisiae. The purified protein kinases were divided into different types according to their elution profiles from the DEAE-cellulose matrix. Two types of cAMP-dependent and two types of cAMP-independent protein kinases were isolated from the wild strain. Differences in the activities of the kinases in the mutants showed a close relationship to the locus of the respective mutations in the cell-cycle. Some properties of the protein kinases are discussed with respect to individual mutations.

Protein kinases control many cellular processes via the ATP-dependent phosphorylation of specific amino acids on target proteins. Despite the availability of the three-dimensional structures of a variety of protein kinases, it has been particularly difficult to explain how noncatalytic domains removed from the active site regulate catalytic function. In this review, we describe how solution methodologies complement the available structural data and help explain how protein kinases may utilize medium-to-long-range effects to regulate substrate phosphorylation. For illustration, two protein kinases, cAMP-dependent protein kinase and the C-terminal Src kinase, are presented as paradigms for the serine/threonine- and tyrosine-specific families. While active-site residues provide an optimal environment for fast phosphoryl group transfer in these and other kinases, the overall rate of protein phosphorylation is limited by nucleotide binding and associated structural changes. Hydrogen-deuterium exchange studies reveal that nucleotide binding induces changes that radiate from a central structural assembly composed of the catalytic loop, glycine-rich loop, and helix alpha C to unique peripheral regions inside and outside the kinase core. This collection of conserved and unique elements delivers information from the active site to distal regions and possibly provides information flow back to the active site. This "push-pull" hypothesis offers a means for understanding how protein kinases can be regulated by protein-protein interactions far from the active site.

Protein kinases play multiple roles in cells and act as the primary messenger enzymes that coordinate cellular responses to the environment. Keywords: protein phosphorylation; apoptosis; gene expression; transcription; disease

A regulatory cascade is a system of signal-transducing proteins that relays signals from receptors to the proteins that carry out a cell's response. Cascades can amplify, integrate, filter and sharpen the signals they transmit. Keywords: signal transduction; protein phosphorylation; protein kinases; amplification; sensitivity