Wynne, Kieran

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Wynne, Kieran
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Wynne, Kieran

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  • Publication
    Cyclic Nucleotide-dependent Protein Kinases Target ARHGAP17 and ARHGEF6 Complexes in Platelets
    (American Society for Biochemistry and Molecular Biology, 2015-12-11)
    Nagy, Zoltan 
    ;
    Wynne, Kieran 
    ;
    Kriegsheim, Alexander von 
    ;
    Gambaryan, Stepan 
    ;
    Smolenski, Albert P. 
    Endothelial cells release prostacyclin (PGI2) and nitric oxide (NO) to inhibit platelet functions. PGI2 and NO effects are mediated by cyclic nucleotides, cAMP- and cGMP-dependent protein kinases (PKA, PKG), and largely unknown PKA and PKG substrate proteins. The small G-protein Rac1 plays a key role in platelets and was suggested to be a target of cyclic nucleotide signaling. We confirm that PKA and PKG activation reduces Rac1-GTP levels. Screening for potential mediators of this effect resulted in the identification of the Rac1-specific GTPase-activating protein ARHGAP17 and the guanine nucleotide exchange factor ARHGEF6 as new PKA and PKG substrates in platelets. We mapped the PKA/PKG phosphorylation sites to serine 702 on ARHGAP17 using Phos-tag gels and to serine 684 on ARHGEF6. We show that ARHGAP17 binds to the actin-regulating CIP4 protein in platelets and that Ser-702 phosphorylation interferes with this interaction. Reduced CIP4 binding results in enhanced inhibition of cell migration by ARHGAP17. Furthermore, we show that ARHGEF6 is constitutively linked to GIT1, a GAP of Arf family small G proteins, and that ARHGEF6 phosphorylation enables binding of the 14-3-3 adaptor protein to the ARHGEF6/GIT1 complex. PKA and PKG induced rearrangement of ARHGAP17- and ARHGEF6-associated protein complexes might contribute to Rac1 regulation and platelet inhibition.
      432Scopus© Citations 22
  • Publication
    Identification of β2-microglobulin as a urinary biomarker for chronic allograft nephropathy using proteomic methods
    (Wiley, 2011-08)
    Johnston, Olwyn 
    ;
    Cassidy, Hilary 
    ;
    O'Connell, Séin 
    ;
    O'Riordan, Aisling 
    ;
    Gallagher, William M. 
    ;
    Maguire, Patricia B. 
    ;
    Wynne, Kieran 
    ;
    Cagney, Gerard 
    ;
    Ryan, Michael P. 
    ;
    Conlon, Peter J. 
    ;
    McMorrow, Tara 
    Chronic allograft nephropathy (CAN) remains the leading cause of renal graft loss after the first year following renal transplantation. This study aimed to identify novel urinary proteomic profiles, which could distinguish and predict CAN in susceptible individuals. Experimental Design: The study included 34 renal transplant patients with histologically proven CAN and 36 patients with normal renal transplant function. High-throughput proteomic profiles were generated from urine samples with three different ProteinChip arrays by surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Following SELDI a biomarker pattern software analysis was performed which led to the identification of a novel biomarker pattern that could distinguish patients with CAN from those with normal renal function. Results: An 11.7 kDa protein identified as β2 microglobulin was the primary protein of this biomarker pattern, distinguishing CAN from control patients (ROC = 0.996). SELDI-TOF-MS comparison of purified β2 microglobulin protein and CAN urine demonstrated identical 11.7 kDa protein peaks. Significantly higher concentrations of β2 microglobulin were found in the urine of patients with CAN compared to the urine of normal renal function transplant recipients (p<0.001). Conclusions and clinical relevance: Whilst further validation in a larger more diverse patient population is required to determine if this β2 microglobulin protein biomarker will provide a potential means of diagnosing CAN by non-invasive methods in a clinical setting, this study clearly shows a capability to stratify control and disease patients.
      968Scopus© Citations 20