Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Wiskott-Aldrich syndrome (WAS) is a rare immunodeficiency disease affecting mainly platelets and lymphocytes. Here, we show that the WAS gene product, WASp, is tyrosine phosphorylated upon aggregation of the high affinity IgE receptor (Fc epsilonRI) at the surface of RBL-2H3 rat tumor mast cells. Lyn and the Bruton's tyrosine kinase (Btk), two protein tyrosine kinases involved in Fc epsilonRI-signaling phosphorylate WASp and interact with WASp in vivo. Interestingly, expression of a GTPase defective mutant form of CDC42, that interacts with WASp, is accompanied by a substantial increase in WASp tyrosine phosphorylation. This study suggests that activated CDC42 recruits WASp to the plasma membrane where it becomes phosphorylated by Lyn and Btk. We conclude that WASp represents a connection between protein tyrosine kinase signaling pathways and CDC42 function in cytoskeleton and cell growth regulation in hematopoietic cells.
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PMID:Tyrosine phosphorylation of the Wiskott-Aldrich syndrome protein by Lyn and Btk is regulated by CDC42. 974 69

Bruton's tyrosine kinase (Btk) has been shown to play a role in normal B-lymphocyte development. Defective expression of Btk leads to human and murine immunodeficiencies. However, the exact role of Btk in the cytoplasmic signal transduction in B cells is still unclear. This study represents a search for the substrate for Btk in vivo. We identified one of the major phosphoproteins associated with Btk in the preB cell line NALM6 as the Wiskott-Aldrich syndrome protein (WASP), the gene product responsible for Wiskott-Aldrich syndrome, which is another hereditary immunodeficiency with distinct abnormalities in hematopoietic cells. We demonstrated that WASP was transiently tyrosine-phosphorylated after B-cell antigen receptor cross-linking on B cells, suggesting that WASP is located downstream of cytoplasmic tyrosine kinases. An in vivo reconstitution system demonstrated that WASP is physically associated with Btk and can serve as the substrate for Btk. A protein binding assay suggested that the tyrosine-phosphorylation of WASP alters the association between WASP and a cellular protein. Furthermore, identification of the phosphorylation site of WASP in reconstituted cells allowed us to evaluate the catalytic specificity of Btk, the exact nature of which is still unknown.
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PMID:Involvement of wiskott-aldrich syndrome protein in B-cell cytoplasmic tyrosine kinase pathway. 1006 73

The Wiskott-Aldrich syndrome protein (WASp) has been implicated in modulation of lymphocyte activation and cytoskeletal reorganization. To address the mechanisms whereby WASp subserves such functions, we have examined WASp roles in lymphocyte development and activation using mice carrying a WAS null allele (WAS(-)(/)(-)). Enumeration of hemopoietic cells in these animals revealed total numbers of thymocytes, peripheral B and T lymphocytes, and platelets to be significantly diminished relative to wild-type mice. In the thymus, this abnormality was associated with impaired progression from the CD44(-)CD25(+) to the CD44(-)CD25(-) stage of differentiation. WASp-deficient thymocytes and T cells also exhibited impaired proliferation and interleukin (IL)-2 production in response to T cell antigen receptor (TCR) stimulation, but proliferated normally in response to phorbol ester/ionomycin. This defect in TCR signaling was associated with a reduction in TCR-evoked upregulation of the early activation marker CD69 and in TCR-triggered apoptosis. While induction of TCR-zeta, ZAP70, and total protein tyrosine phosphorylation as well as mitogen-activated protein kinase (MAPK) and stress-activated protein/c-Jun NH(2)-terminal kinase (SAPK/JNK) activation appeared normal in TCR-stimulated WAS(-)(/)(-) cells, TCR-evoked increases in intracellular calcium concentration were decreased in WASp-deficient relative to wild-type cells. WAS(-)(/)(-) lymphocytes also manifested a marked reduction in actin polymerization and both antigen receptor capping and endocytosis after TCR stimulation, whereas WAS(-)(/)(-) neutrophils exhibited reduced phagocytic activity. Together, these results provide evidence of roles for WASp in driving lymphocyte development, as well as in the translation of antigen receptor stimulation to proliferative or apoptotic responses, cytokine production, and cytoskeletal rearrangement. The data also reveal a role for WASp in modulating endocytosis and phagocytosis and, accordingly, suggest that the immune deficit conferred by WASp deficiency reflects the disruption of a broad range of cellular behaviors.
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PMID:Antigen receptor-induced activation and cytoskeletal rearrangement are impaired in Wiskott-Aldrich syndrome protein-deficient lymphocytes. 1054 4

IMP dehydrogenase is a rate-limiting enzyme involved in the synthesis of GTP. In mammalian cells it is regulated with respect to growth rate and is the target of numerous therapeutic agents. Mutations in the RNA polymerase II elongation machinery render yeast sensitive to inhibitors of IMP dehydrogenase and defective in inducing transcription of one of the IMP dehydrogenase-encoding genes, IMD2. Here we show that loss of IMD2, but not IMD1, IMD3, or IMD4, conferred upon yeast the same drug sensitivity found in elongation mutants. We tested whether the drug sensitivity of elongation mutants is due to their inability to induce IMD2 by providing them with exogenous copies of the gene. In some elongation mutants, overexpression reversed drug sensitivity and a transcriptional defect. Overexpression in mutants with a more severe phenotype partially suppressed drug sensitivity but was inconsequential in reversing a defect in transcription. These findings suggest that the drug sensitivity of elongation mutants is largely but not solely attributable to defects in the ability to induce IMD2, because transcription is compromised even when IMD2 mRNA levels are adequate. We describe two DNA sequence elements in the promoter of the gene that regulate it. We also found that IMD2 mRNA abundance is coupled to cell growth rate. These findings show that yeast possess a conserved system that gauges nucleotide pools and cell growth rate and responds through a uniquely regulated member of the IMD gene family.
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PMID:Regulation of an IMP dehydrogenase gene and its overexpression in drug-sensitive transcription elongation mutants of yeast. 1144 Oct 18

IMP dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo synthesis of GTP. Yeast with mutations in the transcription elongation machinery are sensitive to inhibitors of this enzyme such as 6-azauracil and mycophenolic acid, at least partly because of their inability to transcriptionally induce IMPDH. To understand the molecular basis of this drug-sensitive phenotype, we have dissected the expression and function of a four-gene family in yeast called IMD1 through IMD4. We show here that these family members are distinct, despite a high degree of amino acid identity between the proteins they encode. Extrachromosomal copies of IMD1, IMD3, or IMD4 could not rescue the drug-sensitive phenotype of IMD2 deletants. When overexpressed, IMD3 or IMD4 weakly compensated for deletion of IMD2. IMD1 is transcriptionally silent and bears critical amino acid substitutions compared with IMD2 that destroy its function, offering strong evidence that it is a pseudogene. The simultaneous deletion of all four IMD genes was lethal unless growth media were supplemented with guanine. This suggests that there are no other essential functions of the IMPDH homologs aside from IMP dehydrogenase activity. Although neither IMD3 nor IMD4 could confer drug resistance to cells lacking IMD2, either alone was sufficient to confer guanine prototrophy. The special function of IMD2 was provided by its ability to be transcriptionally induced and the probable intrinsic drug resistance of its enzymatic activity.
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PMID:Functional distinctions between IMP dehydrogenase genes in providing mycophenolate resistance and guanine prototrophy to yeast. 1274 40

IMP dehydrogenase (IMPDH) is the rate-limiting enzyme for de novo GMP synthesis. Its activity is correlated with cell growth, and it is the target of a number of proven and experimental drug therapies including mycophenolic acid (MPA). MPA inhibits the enzyme by trapping a covalent nucleotide-enzyme intermediate. Saccharomyces cerevisiae has four IMPDH genes called IMD1-IMD4. IMD2 is transcriptionally regulated and is the only one that enables yeast to grow in the presence of MPA. We show here that de novo synthesis of the IMD2-encoded protein is strongly induced upon MPA treatment. We also monitor the in vivo formation of a covalent nucleotide-enzyme intermediate for Imd2, Imd3, and Imd4 that accumulates in the presence of MPA. Complete formation of the Imd2 intermediate requires drug concentrations manyfold higher than that required to quantitatively trap the Imd3- or Imd4-nucleotide adducts. Purification of the tagged IMD gene products reveals that the family of polypeptides coassemble to form heteromeric IMPDH complexes, suggesting that they form mixed tetramers. These data demonstrate that S. cerevisiae harbor multiple IMPDH enzymes with varying drug sensitivities and offer an assay to monitor the inhibition of IMPDH in living cells. They also suggest that mixed inhibition profiles may result from heteromeric complexes in cell types that contain multiple IMPDH gene products. The mobility shift assay could serve as a tool for the detection of drug-inactivated IMPDH in the cells of patients receiving MPA therapy.
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PMID:Detection of the mycophenolate-inhibited form of IMP dehydrogenase in vivo. 1529 16

Effector functions mediated by NK cells involve cytotoxicity and transcription-dependent production and release of cytokines and chemokines. Although the JAK/STAT pathway mediates lymphokine-induced transcriptional regulation in NK cells, very little is known about transcriptional regulation induced during cell-cell contact. We demonstrate that the Wiskott-Aldrich syndrome protein (WASp) is an important component for integration of signals leading to nuclear translocation of NFAT2 and NF-kappaB (RelA) during cell-cell contact and NKp46-dependent signaling. This WASp function is independent of its known role in F-actin polymerization and cytoskeletal rearrangement. Absence of WASp results in decreased accumulation of calcineurin, WASp-interacting protein, and molecules upstream of calcium mobilization, i.e., activated ZAP70 and phospholipase C-gamma1, in the disorganized NK cell immune synapse. Production of GM-CSF, but not IFN-gamma, is decreased, while natural cytotoxicity of Wiskott-Aldrich syndrome-NK cells is maintained. Our results indicate that WASp independently regulates its dual functions, i.e., actin cytoskeletal remodeling and transcription in NK cells.
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PMID:The Wiskott-Aldrich syndrome protein regulates nuclear translocation of NFAT2 and NF-kappa B (RelA) independently of its role in filamentous actin polymerization and actin cytoskeletal rearrangement. 1572 66

IMP dehydrogenase (IMPDH) is required for the de novo synthesis of guanine nucleotides. While most invertebrates have one IMPDH gene and humans and mice have two, Saccharomyces cerevisiae contains four, IMD1-IMD4. Although Imd2 is 92% identical to Imd3, it is the only S. cerevisiae IMPDH that is resistant to mycophenolic acid in vitro and is the only one of the four that supports drug-resistant growth. Thus, S. cerevisiae is unique in possessing two classes of IMPDH enzymes with very different drug susceptibilities. The mycophenolate-sensitive growth phenotype has become an important genetic tool in yeast, particularly as an indicator for mutations in the transcription elongation machinery. Here we exploit the distinct drug sensitivity of these two closely related IMPDH genes to identify the naturally occurring determinants of drug-resistant growth. Using chimeric IMD2-IMD3 genes in a strain null for IMD genes, we show that one of the 39 amino acid differences between these enzymes is responsible for much of its drug resistance. The IMP dehydrogenase activity of purified chimeric Imd3 containing the Imd2 residue at position 253 was eight-fold more resistant than native Imd3. The reciprocal change in Imd2 resulted in a 23-fold loss of resistance. Hence, acquisition of a hydroxyl side-chain at 523 is sufficient to confer a drug-resistant phenotype upon this organism. We identified the major determinant of the functional distinction between IMD genes in this yeast and suggest that selective pressure on this species forced divergence of one member of this gene family toward drug resistance.
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PMID:Dissection of the molecular basis of mycophenolate resistance in Saccharomyces cerevisiae. 1627 36

The activities of different kinases have been correlated to the phosphorylation of Wiscott-Aldrich syndrome protein (WASP) by studies in multiple cell systems. The purpose of this study was to elucidate the regulatory mechanisms involved in WASP phosphorylation and the resulting sealing ring formation in osteoclasts. The phosphorylation state of WASP and WASP-interacting proteins was determined in osteoclasts treated with osteopontin or expressing either constitutively active or kinase-defective Src by adenovirus-mediated delivery. In vitro kinase analysis of WASP immunoprecipitates exhibited phosphorylation of c-Src, PYK2, WASP, protein-tyrosine phosphatase (PTP)-PEST, and Pro-Ser-Thr phosphatase-interacting protein (PSTPIP). Phosphorylation of these proteins was increased in osteopontin-treated and constitutively active Src-expressing osteoclasts. Pulldown analysis with glutathione S-transferase-fused proline-rich regions of PTP-PEST revealed coprecipitation of WASP, PYK2, c-Src, and PSTPIP proteins with the N-terminal region (amino acids 294-497) of PTP-PEST. Similarly, interaction of the same signaling proteins, as well as PTP-PEST, was observed with glutathione S-transferase-fused proline-rich regions of WASP. Furthermore, osteopontin stimulation or constitutively active Src expression resulted in serine phosphorylation and inhibition of WASP-associated PTP-PEST. The inhibition of PTP-PEST was accompanied by an increase in tyrosine phosphorylation of WASP and other associated signaling proteins. Experiments with an inhibitor to phosphatase and small interference RNA to PTP-PEST confirmed the involvement of PTP-PEST in sealing ring formation and bone resorption. WASP, which is identified in the sealing ring of resorbing osteoclasts, also demonstrates colocalization with c-Src, PYK2, PSTPIP, and PTP-PEST in immunostaining analyses. Our findings suggest that both tyrosine kinase(s) and the tyrosine phosphatase PTP-PEST coordinate the formation of the sealing ring and thus the bone-resorbing function of osteoclasts.
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PMID:Phosphorylation of a Wiscott-Aldrich syndrome protein-associated signal complex is critical in osteoclast bone resorption. 1728 76

The molecular mechanisms used by oligodendrocyte precursor cells (OPCs), oligodendrocytes (OLs), and Schwann cells (SCs) to advance processes for motility in the developing nervous system and to ensheath axons at myelination are currently not well defined. Here we demonstrate that OPCs, OLs, and SCs express the major proteins involved in actin polymerization-driven protrusion; these key proteins including F-actin, the Arp2/3 complex, neural-Wiskott Aldrich Syndrome protein (N-WASP) and WAVE proteins, and the RhoGTPases Rac and Cdc42 are present at the leading edges of processes being extended by OPCs, OLs, and SCs. We reveal by real-time PCR that OLs and SCs have different dominant WAVE isoforms. Inhibition of the WASP/WAVE protein, N-WASP, with wiskostatin that prevents activation of the Arp2/3 complex, blocks process extension by OPCs and SCs. Inhibition of N-WASP also causes OPC and SC process retraction, which is preceded by retraction of filopodia. This implicates filopodia in OPC and SC process stability and also of N-WASP in OPC and SC process dynamics. We also demonstrate that p34 (a component of the Arp2/3 complex), WASP/WAVE proteins, actin, alpha-tubulin, Rac, Cdc42, vinculin, and focal adhesion kinase are detected in water-shocked myelin purified from brain. Inhibition of N-WASP with wiskostatin decreases the number of axons undergoing initial ensheathment in intact optic nerve samples and reduces the Po content of dorsal root ganglia:SC co-cultures. Our findings indicate that OPCs, OLs, and SCs extend processes using actin polymerization-driven protrusion dependent on N-WASP. We hypothesize that inner mesaxons of OLs and SCs use the same mechanism to ensheath axons at myelination.
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PMID:N-WASP regulates extension of filopodia and processes by oligodendrocyte progenitors, oligodendrocytes, and Schwann cells-implications for axon ensheathment at myelination. 1740 46


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