The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.

Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revealed remarkable similarities, a genetic requirement for MAP kinases in RTK signaling has not been established. During retinal development in Drosophila, the sevenless (Sev) RTK is required for development of the R7 photoreceptor cell. Components of the signal transduction pathway activated by Sev in the R7 precursor include proteins encoded by the gap1, drk, Sos, ras1 and raf loci. In this report we present evidence that a Drosophila MAP kinase, ERK-A, is encoded by the rolled locus and is required downstream of raf in the Sev signal transduction pathway.     

PSK, a novel STE20-like kinase derived from prostatic carcinoma that activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and regulates actin cytoskeletal organization

Degenerate polymerase chain reaction against conserved kinase catalytic subdomains identified 15 tyrosine and serine-threonine kinases expressed in surgically removed prostatic carcinoma tissues, including six receptor kinases (PDGFBR, IGF1-R, VEGFR2, MET, RYK, and EPH-A1), six non-receptor kinases (ABL, JAK1, JAK2, TYK2, PLK-1, and EMK), and three novel kinases. Several of these kinases are oncogenic, and may function in the development of prostate cancer. One of the novel kinases is a new member of the sterile 20 (STE20) family of serine-threonine kinases which we have called prostate-derived STE20-like kinase (PSK) and characterized functionally. PSK encodes an open reading frame of 3705 nucleotides and contains an N-terminal kinase domain. Immunoprecipitated PSK phosphorylates myelin basic protein and transfected PSK stimulates MKK4 and MKK7 and activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway. Microinjection of PSK into cells results in localization of PSK to a vesicular compartment and causes a marked reduction in actin stress fibers. In contrast, C-terminally truncated PSK (1-349) did not localize to this compartment or induce a decrease in stress fibers demonstrating a requirement for the C terminus. Kinase-defective PSK (K57A) was unable to reduce stress fibers. PSK is the first member of the STE20 family lacking a Cdc42/Rac binding domain that has been shown to regulate both the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and the actin cytoskeleton.     

Dehydroepiandrosterone negatively regulates the p38 mitogen-activated protein kinase pathway by a novel mitogen-activated protein kinase phosphatase

Dehydroepiandrosterone-sulfate, the sulfated form of dehydroepiandrosterone, is the most abundant steroid in young adults, but gradually declines with aging. In humans, the clinical application of dehydroepiandrosterone targeting some collagen diseases, such as systemic lupus erythematosus, as an adjunctive treatment has been applied in clinical trial. Here, we report that dehydroepiandrosterone may negatively regulate the mitogen-activated protein kinase pathway in humans via a novel dual specificity protein phosphatase, DDSP (dehydroepiandrosterone-enhanced dual specificity protein phosphatase). DDSP is highly homologous to LCPTP/HePTP, a tissue-specific protein tyrosine phosphatase (PTP) which negatively regulates both ERK and p38-mitogen-activated protein kinase, and is transcribed from the PTPN7 locus by alternative splicing. Although previous reports have shown that the mRNA expression of the LCPTP/HePTP gene was inducible by extracellular signals such as T-cell antigen receptor stimulation, reverse transcribed (RT)-PCR experiments using specific sets of primers suggested that the expression of LCPTP/HePTP was constitutive while the actual inducible sequence was that of DDSP. Furthermore DDSP was widely distributed among different types of human tissues and specifically interacted with p38-mitogen-activated protein kinase. This inducible negative regulation of the p38-mitogen-activated protein kinase-dependent pathway may help to clarify the broad range of dehydroepiandrosterone actions, thereby aiding the development of new preventive or adjunctive applications for human diseases.     

CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis

Plants respond to environmental stress by activating "stress genes." The plant hormone abscisic acid (ABA) plays an important role in stress-responsive gene expression. Although Ca(2+) serves as a common second messenger in signaling stress and ABA, little is known about the molecular basis of Ca(2+) action in these pathways. Here, we show that CIPK3, a Ser/Thr protein kinase that associates with a calcineurin B-like calcium sensor, regulates ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis. The expression of the CIPK3 gene itself is responsive to ABA and stress conditions, including cold, high salt, wounding, and drought. Disruption of CIPK3 altered the expression pattern of a number of stress gene markers in response to ABA, cold, and high salt. However, drought-induced gene expression was not altered in the cipk3 mutant plants, suggesting that CIPK3 regulates select pathways in response to abiotic stress and ABA. These results identify CIPK3 as a molecular link between stress- and ABA-induced calcium signal and gene expression in plant cells. Because the cold signaling pathway is largely independent of endogenous ABA production, CIPK3 represents a cross-talk "node" between the ABA-dependent and ABA-independent pathways in stress responses.     

The balance between the novel protein target of wingless and the Drosophila Rho-associated kinase pathway regulates planar cell polarity in the Drosophila wing

Planar cell polarity (PCP) signaling is mediated by the serpentine receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling utilizes Drosophila Frizzled 2 (DFz2) as a receptor and also requires Dsh for transducing signals to regulate cell proliferation and differentiation in many developmental contexts. Distinct pathways are activated downstream of Dsh in Wg- and Fz-signaling pathways. Recently, a number of genes, which have essential roles as downstream components of PCP signaling, have been identified in Drosophila. They include the small GTPase RhoA/Rho1, its downstream effector Drosophila rho-associated kinase (Drok), and a number of genes such as inturned (in) and fuzzy (fy), whose biochemical functions are unclear. RhoA and Drok provide a link from Fz/Dsh signaling to the modulation of actin cytoskeleton. Here we report the identification of the novel gene target of wingless (tow) by enhancer trap screening. tow expression is negatively regulated by Wg signaling in wing imaginal discs, and the balance between tow and the Drok pathway regulates wing-hair morphogenesis. A loss-of-function mutation in tow does not result in a distinct phenotype. Genetic interaction and gain-of-function studies provide evidence that Tow acts downstream of Fz/Dsh and plays a role in restricting the number of hairs that wing cells form.     

Protein kinase D (PKD) activation in intact cells through a protein kinase C-dependent signal transduction pathway.

Protein kinase D (PKD) is a serine/threonine protein kinase that is directly stimulated in vitro by phorbol esters and diacylglycerol in the presence of phospholipids. Here, we examine the regulation of PKD in living cells. Our results demonstrate that tumour-promoting phorbol esters, membrane-permeant diacylglycerol and serum growth factors rapidly induced PKD activation in immortalized cell lines (e.g. Swiss 3T3 and Rat-1 cells), in secondary cultures of mouse embryo fibroblasts and in COS-7 cells transiently transfected with a PKD expression construct. PKD activation was maintained during cell disruption and immunopurification and was associated with an electrophoretic mobility shift and enhanced 32P incorporation into the enzyme, but was reversed by treatment with alkaline phosphatase. PKD was activated, deactivated and reactivated in response to consecutive cycles of addition and removal of PDB. PKD activation was completely abrogated by exposure of the cells to the protein kinase C inhibitors GF I and Ro 31-8220. In contrast, these compounds did not inhibit PKD activity when added directly in vitro. Co-transfection of PKD with constitutively activated mutants of PKCs showed that PKCepsilon and eta but not PKCzeta strongly induced PKD activation in COS-7 cells. Thus, our results indicate that PKD is activated in living cells through a PKC-dependent signal transduction pathway.     

Insulin signal transduction through protein kinase cascades

This review summarizes the evolution of ideas concerning insulin signal transduction, the current information on protein ser/thr kinase cascades as signalling intermediates, and their status as participants in insulin regulation of energy metabolism. Best characterized is the Ras-MAPK pathway, whose input is crucial to cell fate decisions, but relatively dispensable in metabolic regulation. By contrast the effectors downstream of PI-3 kinase, although less well elucidated, include elements indispensable for the insulin regulation of glucose transport, glycogen and cAMP metabolism. Considerable information has accrued on PKB/cAkt, a protein kinase that interacts directly with Ptd Ins 3OH phosphorylated lipids, as well as some of the elements further downstream, such as glycogen synthase kinase-3 and the p70 S6 kinase. Finally, some information implicates other erk pathways (e.g. such as the SAPK/JNK pathway) and Nck/cdc42-regulated PAKs (homologs of the yeast Ste 20) as participants in the cellular response to insulin. Thus insulin recruits a broad array of protein (ser/thr) kinases in its target cells to effectuate its characteristic anabolic and anticatabolic programs.     

Oxalate selectively activates p38 mitogen-activated protein kinase and c-Jun N-terminal kinase signal transduction pathways in renal epithelial cells

Oxalate, a metabolic end product, is an important factor in the pathogenesis of renal stone disease. Oxalate exposure to renal epithelial cells results in re-initiation of the DNA synthesis, altered gene expression, and apoptosis, but the signaling pathways involved in these diverse effects have not been evaluated. The effects of oxalate on mitogen- and stress-activated protein kinase signaling pathways were studied in LLC-PK1 cells. Exposure to oxalate (1 mM) rapidly stimulated robust phosphorylation and activation of p38 MAPK. Oxalate exposure also induced modest activation of JNK, as monitored by phosphorylation of c-Jun. In contrast, oxalate exposure had no effect on phosphorylation and enzyme activity of p42/44 MAPK. We also show that specific inhibition of p38 MAPK by 4(4-(fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl)imidazole (SB203580) or by overexpression of a kinase-dead dominant negative mutant of p38 MAPK abolishes oxalate induced re-initiation of DNA synthesis in LLC-PK1 cells. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as MAPK-activated protein kinase-2 activity in cell extracts. Thus, this study not only provides the first demonstration of selective activation of p38 MAPK and JNK signaling pathways by oxalate but also suggests that p38 MAPK activity is essential for the effects of oxalate on re-initiation of DNA synthesis.     

COM crystals activate the p38 mitogen-activated protein kinase signal transduction pathway in renal epithelial cells

Interaction of calcium oxalate monohydrate (COM) crystals with renal cells has been shown to result in altered gene expression, DNA synthesis, and cell death. In the current study the role of a stress-specific p38 MAP kinase-signaling pathway in mediating these effects of COM crystals was investigated. Exposure of cells to COM crystals (20 microg/cm(2)) rapidly stimulated strong phosphorylation and activation of p38 mitogen-activated protein kinase (p38 MAP kinase) and re-initiation of DNA synthesis. Inhibition of COM crystal binding to the cells by heparin blocked the effects of COM crystals on p38 MAPK activation. We also show that specific inhibition of p38 MAPK by 4-(4-fluorophenyl)-2-(4-methylsulfonylphenyl)-5-(4-pyridyl) imidazole (SB203580) or by overexpression of a dominant negative mutant of p38 MAP kinase abolishes COM crystal-induced re-initiation of DNA synthesis. The inhibition is dose-dependent and correlates with in situ activity of native p38 MAP kinase, determined as mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP kinase-2) activity in cell extracts. In summary, inhibiting activation of p38 MAPK pathway abrogated the DNA synthesis in response to COM crystals. These data are the first demonstrations of activation of the p38 MAPK signaling pathway by COM crystals and suggest that, in response to COM crystals, this pathway transduces critical signals governing the re-initiation of DNA synthesis in renal epithelial cells.     

The MAP kinase cascade. Discovery of a new signal transduction pathway

Using biochemical techniques similar to those used by Krebs and Fischer in elucidating the cAMP kinase cascade, a protein kinase cascade has been found that represents a new pathway for signal transduction. This pathway is activated in almost all cells that have been examined by many different growth and differentiations factors suggesting control of different cell responses. At this writing, four tiers of growth factor regulated kinases, each tier represented by more than one enzyme, have been reconstitutedin vitro to form the MAP kinase cascade. Preliminary findings suggesting multiple feedback or feedforward regulation of several components in the cascade predict higher complexity than a simple linear pathway.