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ABSTRACT: Soluble guanylyl cyclase (sGC) is the major cytosolic receptor for nitric oxide (NO) that converts GTP into the second messenger cGMP in a NO-dependent manner. Other factors controlling this key enzyme are intracellular proteins such as Hsp90 and PSD95, which bind to sGC and modulate its activity, stability, and localization. To date little is known about the effects of posttranslational modifications of sGC, although circumstantial evidence suggests that reversible phosphorylation may contribute to sGC regulation. Here we demonstrate that inhibitors of protein-tyrosine phosphatases such as pervanadate and bisperoxo(1,10-phenanthroline)oxovanadate(V) as well as reactive oxygen species such as H2O2 induce specific tyrosine phosphorylation of the beta1 but not of the alpha1 subunit of sGC. Tyrosine phosphorylation of sGCbeta1 is also inducible by pervanadate and H2O2 in intact PC12 cells, rat aortic smooth muscle cells, and in rat aortic tissues, indicating that tyrosine phosphorylation of sGC may also occur in vivo. We have mapped the major tyrosine phosphorylation site to position 192 of beta1, where it forms part of a highly acidic phospho-acceptor site for Src-like kinases. In the phosphorylated state Tyr(P)-192 exposes a docking site for SH2 domains and efficiently recruits Src and Fyn to sGCbeta1, thereby promoting multiple phosphorylation of the enzyme. Our results demonstrate that sGC is subject to tyrosine phosphorylation and interaction with Src-like kinases, revealing an unexpected cross-talk between the NO/cGMP and tyrosine kinase signaling pathways at the level of sGC.
Journal of Biological Chemistry 10/2005; 280(39):33149-56. · 4.77 Impact Factor
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ABSTRACT: Nitric oxide (NO) plays a key role in vascular function, cell proliferation, and apoptosis. Proper subcellular localization of endothelial NO synthase (eNOS) is crucial for its activity; however, the role of eNOS trafficking for NO biosynthesis remains to be defined. Overexpression of NOS-interacting protein (NOSIP) induces translocation of eNOS from the plasma membrane to intracellular compartments, thereby impairing NO production. Here we report that endogenous NOSIP reduces the enzymatic capacity of eNOS, specifically in the G(2) phase of the cell cycle by targeting eNOS to the actin cytoskeleton. This regulation is critically dependent on the nucleocytoplasmic shuttling of NOSIP and its cytoplasmic accumulation in the G(2) phase. The predominant nuclear localization of NOSIP depends on a bipartite nuclear localization sequence (NLS) mediating interaction with importin alpha. Mutational destruction of the NLS abolishes nuclear import and interaction with importin alpha. Nuclear export is insensitive to leptomycin B and hence different from the CRM1-dependent default mechanism. Inhibition of NOSIP expression by RNA interference completely abolishes G(2)-specific cytoskeletal association and inhibition of eNOS. These findings describe a novel cell cycle-dependent modulation of endogenous NO levels that are critical to the cell cycle-related actions of NO such as apoptosis or cell proliferation.
Molecular and Cellular Biology 10/2005; 25(18):8251-8. · 5.53 Impact Factor
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BMC Pharmacology. 01/2005;
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BMC Pharmacology. 01/2005;
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BMC Pharmacology. 01/2005;
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BMC Pharmacology. 01/2005;
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ABSTRACT: Nitric oxide (NO)-sensitive soluble guanylyl cyclase (sGC) is the major cytosolic receptor for NO, catalyzing the conversion of GTP to cGMP. In a search for proteins specifically interacting with human sGC, we have identified the multidomain protein AGAP1, the prototype of an ArfGAP protein with a GTPase-like domain, Ankyrin repeats, and a pleckstrin homology domain. AGAP1 binds through its carboxyl terminal portion to both the alpha1 and beta1 subunits of sGC. We demonstrate that AGAP1 mRNA and protein are co-expressed with sGC in human, murine, and rat cells and tissues and that the two proteins interact in vitro and in vivo. We also show that AGAP1 is prone to tyrosine phosphorylation by Src-like kinases and that tyrosine phosphorylation potently increases the interaction between AGAP1 and sGC, indicating that complex formation is modulated by reversible phosphorylation. Our findings may hint to a potential role of AGAP1 in integrating signals from Arf, NO/cGMP, and tyrosine kinase signaling pathways.
Journal of Biological Chemistry 12/2004; 279(47):49346-54. · 4.77 Impact Factor
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ABSTRACT: Insulinoma-associated protein (IA)-2beta, also known as phogrin, is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase family and is located in dense-core secretory vesicles. In patients with type 1 diabetes, autoantibodies to IA-2beta appear years before the development of clinical disease. The genomic structure and function of IA-2beta, however, is not known. In the present study, we determined the genomic structure of IA-2beta and found that both human and mouse IA-2beta consist of 23 exons and span approximately 1,000 and 800 kb, respectively. With this information, we prepared a targeting construct and inactivated the mouse IA-2beta gene as demonstrated by lack of IA-2beta mRNA and protein expression. The IA-2beta(-/-) mice, in contrast to wild-type controls, showed mild glucose intolerance and impaired glucose-stimulated insulin secretion. Knockout of the IA-2beta gene in NOD mice, the most widely studied animal model for human type 1 diabetes, failed to prevent the development of cyclophosphamide-induced diabetes. We conclude that IA-2beta is involved in insulin secretion, but despite its importance as a major autoantigen in human type 1 diabetes, it is not required for the development of diabetes in NOD mice.
Diabetes 08/2004; 53(7):1684-91. · 8.29 Impact Factor
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ABSTRACT: Soluble guanylyl cyclase (sGC) is a ubiquitous enzyme that functions as a receptor for nitric oxide. Despite the obligate heterodimeric nature of sGC, the sequence segments mediating subunit association have remained elusive. Our initial screening for relevant interaction site(s) in the most common sGC isoenzyme, alpha(1) beta(1), identified two regions in each subunit, i.e. the regulatory domains and the central regions, contributing to heterodimer formation. To map the relevant segments in the beta(1) subunit precisely, we constructed multiple N- and C-terminal deletion variants and cotransfected them with full-length alpha(1) in COS cells. Immunoprecipitation revealed that a sequence segment spanning positions 204-408 mediates binding of beta(1) to alpha(1) The same region of beta(1)[204-408] was found to promote beta /beta(1) homodimerization. Fusion of [204 beta(1)-408] to enhanced green fluorescent protein conferred binding activity to the recipient protein. Coexpression of beta(1)[204-408] with alpha(1) or beta(1) targeted the sGC subunits for proteasomal degradation, suggesting that beta(1)[204-408] forms structurally deficient complexes with alpha(1) and beta(1). Analysis of deletion constructs lacking portions of the beta(1) dimerization region identified two distinct segments contributing to alpha(1) binding, i.e. an N-terminal site covering positions 204-244 and a C-terminal site at 379-408. Both sites are crucial for sGC function because deletion of either site rendered sGC dimerization-deficient and thus functionally inactive. We conclude that the dimerization region of beta(1) extends over 205 residues of its regulatory and central domains and that two discontinuous sites of 41 and 30 residues, respectively, facilitate binding of beta(1) to the alpha(1) subunit of sGC.
Journal of Biological Chemistry 07/2004; 279(24):24935-43. · 4.77 Impact Factor
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ABSTRACT: Most receptor-type protein-tyrosine phosphatases (RPTPs) contain two tandem PTP domains. For some RPTPs the enzymatically inactive membrane-distal phosphatase domains (D2) were found to bind enzymatically active membrane proximal PTP (D1) domains, and oligomerization has been proposed as a general regulatory mechanism. The RPTP-like proteins IA-2 and IA-2beta, major autoantigens in insulin-dependent diabetes mellitus, contain just a single enzymatically inactive PTP-like domain. Their physiological role is as yet enigmatic. To investigate whether the catalytically inactive cytoplasmic domains of IA-2 and IA-2beta are involved in oligomerization, we exploited interaction trap assay in yeast and glutathione S-transferase pull-down and co-immunoprecipitation strategies on lysates of transfected COS-1 cells. The results show that IA-2 and IA-2beta are capable of homo- and heterodimerization to which both the juxtamembrane region and the phosphatase-like segment can contribute. Furthermore, they can form heterodimers with some other RPTP members, most notably RPTPalpha and RPTPepsilon, and down-regulate RPTPalpha enzymatic activity. Thus, in addition to homo-dimerization, the enzymatic activity of receptor-type PTPs can be regulated through heterodimerization with other RPTPs, including the catalytically inactive IA-2 and IA-2beta.
Journal of Biological Chemistry 01/2003; 277(50):48139-45. · 4.77 Impact Factor
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[show abstract]
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ABSTRACT: Most receptor-type protein-tyrosine phosphatases (RPTPs) contain two tandem PTP domains. For some RPTPs the enzymatically
inactive membrane-distal phosphatase domains (D2) were found to bind enzymatically active membrane proximal PTP (D1) domains,
and oligomerization has been proposed as a general regulatory mechanism. The RPTP-like proteins IA-2 and IA-2β, major autoantigens
in insulin-dependent diabetes mellitus, contain just a single enzymatically inactive PTP-like domain. Their physiological
role is as yet enigmatic. To investigate whether the catalytically inactive cytoplasmic domains of IA-2 and IA-2β are involved
in oligomerization, we exploited interaction trap assay in yeast and glutathione S-transferase pull-down and co-immunoprecipitation strategies on lysates of transfected COS-1 cells. The results show that
IA-2 and IA-2β are capable of homo- and heterodimerization to which both the juxtamembrane region and the phosphatase-like
segment can contribute. Furthermore, they can form heterodimers with some other RPTP members, most notably RPTPα and RPTPε,
and down-regulate RPTPα enzymatic activity. Thus, in addition to homo-dimerization, the enzymatic activity of receptor-type
PTPs can be regulated through heterodimerization with other RPTPs, including the catalytically inactive IA-2 and IA-2β.
Journal of Biological Chemistry 12/2002; 277(50):48139-48145. · 4.77 Impact Factor
