Myosin light chain kinase (MLCK) gene disruption in Dictyostelium: a role for MLCK-A in cytokinesis and evidence for multiple MLCKs.

We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A-cells are able, however, to undergo development and to cap crosslinked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A-cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A-cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the other MLCK(s) in the cell.

Urea-inducible Egr-1 transcription in renal inner medullary collecting duct (mIMCD3) cells is mediated by extracellular signal-regulated kinase activation.

Urea (200-400 milliosmolar) activates transcription, translation of, and trans-activation by the immediate-early gene transcription factor Egr-1 in a renal epithelial cell-specific fashion. The effect at the transcriptional level has been attributed to multiple serum response elements and their adjacent Ets motifs located within the Egr-1 promoter. Elk-1, a principal ternary complex factor and Ets domain-containing protein, is a substrate of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. In the renal medullary mIMCD3 cell line, urea (200-400 milliosmolar) activated both ERK1 and ERK2 as determined by in-gel kinase assay and immune-complex kinase assay of epitope-tagged] ERK1 and ERK2. Importantly, urea did not affect abundance of either ERK. Urea-inducible Egr-1 transcription was a consequence of ERK activation because the ERK-specific inhibitor, PD98059, abrogated transcription from the murine Egr-1 promoter in a luciferase reported gene assay. In addition, activators of protein kinase A, including forskolin and 8-Br-cAMP, which are known to inhibit ERK-mediated events, also inhibited urea-inducible Egr-1 transcription. Furthermore, urea-inducible activation of the physiological ERK substrate and transcription factor, Elk-1, was demonstrated through transient cotransfection of a chimeric Elk-1/GAL4 expression plasmid and a GAL4-driven luciferase reporter plasmid. Taken together, these data indicate that, in mIMCD3 cells, urea activates ERKs and the ERK substrate, Elk-1, and that ERK inhibition abrogates urea-inducible Egr-1 transcription. These data are consistent with a model of urea-inducible renal medullary gene expression wherein sequential activation of ERKs and Elk-1 results in increased transcription of Egr-1 through serum response element/Ets motifs.

A molecular mechanism for the effect of lithium on development.

Lithium, one of the most effective drugs for the treatment of bipolar (manic-depressive) disorder, also has dramatic effects on morphogenesis in the early development of numerous organisms. How lithium exerts these diverse effects is unclear, but the favored hypothesis is that lithium acts through inhibition of inositol monophosphatase (IMPase). We show here that complete inhibition of IMPase has no effect on the morphogenesis of Xenopus embryos and present a different hypothesis to explain the broad action of lithium. Our results suggest that lithium acts through inhibition of glycogen synthase kinase-3 beta (GSK-3 beta), which regulates cell fate determination in diverse organisms including Dictyostelium, Drosophila, and Xenopus. Lithium potently inhibits GSK-3 beta activity (Ki = 2 mM), but is not a general inhibitor of other protein kinases. In support of this hypothesis, lithium treatment phenocopies loss of GSK-3 beta function in Xenopus and Dictyostelium. These observations help explain the effect of lithium on cell-fate determination and could provide insights into the pathogenesis and treatment of bipolar disorder.

Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport.

The transferred DNA (T-DNA) of Agrobacterium tumefaciens serves as an insertional mutagen once integrated into a host plant's genome. As a means of facilitating reverse genetic analysis in Arabidopsis thaliana, we have developed a method that allows one to search for plants carrying F-DNA insertions within any sequenced Arabidopsis gene. Using PCR, we screened a collection of 9100 independent T-DNA-transformed Arabidopsis lines and found 17 T-DNA insertions within the 63 genes analyzed. The genes surveyed include members of various gene families involved in signal transduction and ion transport. As an example, data are shown for a T-DNA insertion that was found within CPK-9, a member of the gene family encoding calmodulin-domain protein kinases.

Strong regulation of slow anion channels and abscisic acid signaling in guard cells by phosphorylation and dephosphorylation events.

Recent evidence suggests that slow anion channels in guard cells need to be activated to trigger stomatal closing and efficiently inactivated during stomatal opening. The patch-clamp technique was employed here to determine mechanisms that produce strong regulation of slow anion channels in guard cells. MgATP in guard cells, serving as a donor for phosphorylation, leads to strong activation of slow anion channels. Slow anion-channel activity was almost completely abolished by removal of cytosolic ATP or by the kinase inhibitors K-252a and H7. Nonhydrolyzable ATP, GTP, and guanosine 5'-[gamma-thio]triphosphate did not replace the ATP requirement for anion-channel activation. In addition, down-regulation of slow anion channels by ATP removal was inhibited by the phosphatase inhibitor okadaic acid. Stomatal closures in leaves induced by the plant hormone abscisic acid (ABA) and malate were abolished by kinase inhibitors and/or enhanced by okadaic acid. These data suggest that ABA signal transduction may proceed by activation of protein kinases and inhibition of an okadaic acid-sensitive phosphatase. This modulation of ABA-induced stomatal closing correlated to the large dynamic range for up- and down-regulation of slow anion channels by opposing phosphorylation and dephosphorylation events in guard cells. The presented opposing regulation by kinase and phosphatase modulators could provide important mechanisms for signal transduction by ABA and other stimuli during stomatal movements.

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.

PAK1, a gene that can regulate p53 activity in yeast.

The ability of p53 protein to activate transcription is central to its tumor-suppressor function. We describe a genetic selection in Saccharomyces cerevisiae which was used to isolate a mutant strain defective in p53-mediated transcriptional activation. The defect was partially corrected by overexpression of a yeast gene named PAK1 (p53 activating kinase), which localizes to the left arm of chromosome IX. PAK1 is predicted to encode an 810-aa protein with regions of strong similarity to previously described Ser/Thr-specific protein kinases. PAK1 sequences upstream of the coding region are characteristic of those regulating genes involved in cell cycle control. Expression of PAK1 was associated with an increased specific activity of p53 in DNA-binding assays accompanied by a corresponding increase in transactivation. Thus, PAK1 is the prototype for a class of genes that can regulate the activity of p53 in vivo, and the system described here should be useful in identifying other genes in this class.

Surface signaling in pathogenesis.

Surface signaling plays a major role in fungal infection. Topographical features of the plant surface and chemicals on the surface can trigger germination of fungal spores and differentiation of the germ tubes into appressoria. Ethylene, the fruit-ripening hormone, triggers germination of conidia, branching of hyphae, and multiple appressoria formation in Colletotrichum, thus allowing fungi to time their infection to coincide with ripening of the host. Genes uniquely expressed during appressoria formation induced by topography and surface chemicals have been isolated. Disruption of some of them has been shown to decrease virulence on the hosts. Penetration of the cuticle by the fungus is assisted by fungal cutinase secreted at the penetration structure of the fungus. Disruption of cutinase gene in Fusarium solani pisi drastically decreased its virulence. Small amounts of cutinase carried by spores of virulent pathogens, upon contact with plant surface, release small amounts of cutin monomers that trigger cutinase gene expression. The promoter elements involved in this process in F. solani pisi were identified, and transcription factors that bind these elements were cloned. One of them, cutinase transcription factor 1, expressed in Escherichia coli, is phosphorylated. Several protein kinases from F. solani pisi were cloned. The kinase involved in phosphorylation of specific transcription factors and the precise role of phosphorylation in regulating cutinase gene transcription remain to be elucidated.

The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system.

The simple gas ethylene affects numerous physiological processes in the growth and development of higher plants. With the use of molecular genetic approaches, we are beginning to learn how plants perceive ethylene and how this signal is transduced. Components of ethylene signal transduction are defined by ethylene response mutants in Arabidopsis thaliana. The genes corresponding to two of these mutants, etr1 and etr1, have been cloned. The ETR1 gene encodes a homolog of two-component regulators that are known almost exclusively in prokaryotes. The two-component regulators in prokaryotes are involved in the perception and transduction of a wide range of environmental signals leading to adaptive responses. The CTR1 gene encodes a homolog of the Raf family of serine/threonine protein kinases. Raf is part of a mitogen-activated protein kinase cascade known to regulate cell growth and development in mammals, worms, and flies. The ethylene response pathway may, therefore, exemplify a conserved protein kinase cascade regulated by a two-component system. The dominance of all known mutant alleles of ETR1 may be due to either constitutive activation of the ETR1 protein or dominant interference of wild-type activity. The discovery of Arabidopsis genes encoding proteins related to ETR1 suggests that the failure to recover recessive etr1 mutant alleles may be due to the presence of redundant genes.

Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-1 activation through a pathway that is independent of extracellular signal-regulated kinases, c-Jun N-terminal kinases, and P38 kinase

Solar UV irradiation is the causal factor for the increasing incidence of human skin carcinomas. The activation of the transcription factor activator protein-1 (AP-1) has been shown to be responsible for the tumor promoter action of UV light in mammalian cells. We demonstrate that proteinase inhibitor I (Inh I) and II (Inh II) from potato tubers, when applied to mouse epidermal JB6 cells, block UV-induced AP-1 activation. The inhibition appears to be specific for UV-induced signal transduction for AP-1 activation, because these inhibitors did not block UV-induced p53 activation nor did they exhibit any significant influence on epidermal growth factor-induced AP-1 transactivation. Furthermore, the inhibition of UV-induced AP-1 activity occurs through a pathway that is independent of extracellular signal-regulated kinases and c-Jun N-terminal kinases as well as P38 kinases. Considering the important role of AP-1 in tumor promotion, it is possible that blocking UV-induced AP-1 activity by Inh I or Inh II may be functionally linked to irradiation-induced cell transformation.

Microdomain-dependent Regulation of Lck and Fyn Protein-Tyrosine Kinases in T Lymphocyte Plasma Membranes

Src family protein-tyrosine kinases are implicated in signaling via glycosylphosphatidylinositol (GPI)-anchored receptors. Both kinds of molecules reside in opposite leaflets of the same sphingolipid-enriched microdomains in the lymphocyte plasma membrane without making direct contact. Under detergent-free conditions, we isolated a GPI-enriched plasma membrane fraction, also containing transmembrane proteins, selectively associated with sphingolipid microdomains. Nonionic detergents released the transmembrane proteins, yielding core sphingolipid microdomains, limited amounts of which could also be obtained by detergent-free subcellular fractionation. Protein-tyrosine kinase activity in membranes containing both GPI-anchored and transmembrane proteins was much lower than in core sphingolipid microdomains but was strongly reactivated by nonionic detergents. The inhibitory mechanism acting on Lck and Fyn kinases in these membranes was independent of the protein-tyrosine phosphatase CD45 and was characterized as a mixed, noncompetitive one. We propose that in lymphocyte plasma membranes, Lck and Fyn kinases exhibit optimal activity when juxtaposed to the GPI- and sphingolipid-enriched core microdomains but encounter inhibitory conditions in surrounding membrane areas that are rich in glycerophospholipids and contain additional transmembrane proteins.

Induction of the Tat-binding protein 1 gene accompanies the disabling of oncogenic erbB receptor tyrosine kinases

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic p185neu. We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-p185neu-treated tumor cells but not in cells expressing internalization defective p185neu receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro, and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1. Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

Monoclonal antibodies reveal receptor specificity among G-protein-coupled receptor kinases.

Guanine nucleotide-binding regulatory protein (G protein)-coupled receptor kinases (GRKs) constitute a family of serine/threonine kinases that play a major role in the agonist-induced phosphorylation and desensitization of G-protein-coupled receptors. Herein we describe the generation of monoclonal antibodies (mAbs) that specifically react with GRK2 and GRK3 or with GRK4, GRK5, and GRK6. They are used in several different receptor systems to identify the kinases that are responsible for receptor phosphorylation and desensitization. The ability of these reagents to inhibit GRK- mediated receptor phosphorylation is demonstrated in permeabilized 293 cells that overexpress individual GRKs and the type 1A angiotensin II receptor. We also use this approach to identify the endogenous GRKs that are responsible for the agonist-induced phosphorylation of epitope-tagged beta2- adrenergic receptors (beta2ARs) overexpressed in rabbit ventricular myocytes that are infected with a recombinant adenovirus. In these myocytes, anti-GRK2/3 mAbs inhibit isoproterenol-induced receptor phosphorylation by 77%, while GRK4-6-specific mAbs have no effect. Consistent with the operation of a betaAR kinase-mediated mechanism, GRK2 is identified by immunoblot analysis as well as in a functional assay as the predominant GRK expressed in these cells. Microinjection of GRK2/3-specific mAbs into chicken sensory neurons, which have been shown to express a GRK3-like protein, abolishes desensitization of the alpha2AR-mediated calcium current inhibition. The intracellular inhibition of endogenous GRKs by mAbs represents a novel approach to the study of receptor specificities among GRKs that should be widely applicable to many G-protein-coupled receptors.

Regulation of the retinoblastoma protein-related protein p107 by G1 cyclin-associated kinases.

p107 is a retinoblastoma protein-related phosphoprotein that, when overproduced, displays a growth inhibitory function. It interacts with and modulates the activity of the transcription factor, E2F-4. In addition, p107 physically associates with cyclin E-CDK2 and cyclin A-CDK2 complexes in late G1 and at G1/S, respectively, an indication that cyclin-dependent kinase complexes may regulate, contribute to, and/or benefit from p107 function during the cell cycle. Our results show that p107 phosphorylation begins in mid G1 and proceeds through late G1 and S and that cyclin D-associated kinase(s) contributes to this process. In addition, E2F-4 binds selectively to hypophosphorylated p107, and G1 cyclin-dependent p107 phosphorylation leads to the dissociation of p107-E2F-4 complexes as well as inactivation of p107 G1 blocking function.

Activation of Tsk and Btk tyrosine kinases by G protein beta gamma subunits.

Tsk/Itk and Btk are members of the pleckstrin-homology (PH) domain-containing tyrosine kinase family. The PH domain has been demonstrated to be able to interact with beta gamma subunits of heterotrimeric guanine nucleotide-binding proteins (G proteins) (G beta gamma) and phospholipids. Using cotransfection assays, we show here that the kinase activities of Tsk and Btk are stimulated by certain G beta gamma subunits. Furthermore, using an in vitro reconstitution assay with purified bovine brain G beta gamma subunits and the immunoprecipitated Tsk, we find that Tsk kinase activity is increased by G beta gamma subunits and another membrane factor(s). These results indicate that this family of tyrosine kinases could be an effector of heterotrimeric G proteins.