Browsing by Author "Walsh, Michael P."
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- ItemOpen AccessCalcium-independent smooth muscle contraction: a focus on zipper-interacting protein kinase (ZIPK)(2011) Moffat, Lori Dawn; Walsh, Michael P.; MacDonald, Justin A.
- ItemOpen Access
- ItemOpen AccessIdentification and characterization of caldesmon kinase from chicken gizzard smooth muscle(1988) Scott-Woo, Gisele; Walsh, Michael P.
- ItemOpen AccessIdentification and Functional Characterization of Protein Kinase A-catalyzed Phosphorylation of Potassium Channel Kv1.2 at Serine 449(Journal of Biological Chemistry, 2009-04-22) Johnson, Rosalyn P.; El-Yazbi, Ahmed F.; Hughes, Morgan F.; Schriemer, David C.; Walsh, Emma J.; Walsh, Michael P.; Cole, William C.Vascular smooth muscle Kv1 delayed rectifier K+ channels (KDR) containing Kv1.2 control membrane potential and thereby regulate contractility. Vasodilatory agonists acting via protein kinase A (PKA) enhance vascule smooth muscle Kv1 activity, but the molecular basis of this regulation is uncertain. We characterized the role of a C-terminal phosphorylation site, Ser-449, in Kv1.2 expressed in HEK 293 cells by biochemical and electrophysiological methods. We found that 1) in vitro phosphorylation of Kv1.2 occurred exclusively at serine residues, 2) one major phosphopeptide that co-migrated with 449pSASTISK was generated by proteolysis of in vitro phosphorylated Kv1.2, 3) the peptide 445KKSRSASTISK exhibited stoichiometric phosphorylation by PKA in vitro, 4) matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy (MS) and MS/MS confirmed in vitro Ser-449 phosphorylation by PKA, 5) in situ phosphorylation at Ser-449 was detected in HEK 293 cells by MALDI-TOF MS followed by MS/MS. MIDAS (multiple reaction monitoring-initiated detection and sequencing) analysis revealed additional phosphorylated residues, Ser-440 and Ser-441, 6) in vitro 32P incorporation was significantly reduced in Kv1.2-S449A, Kv1.2-S449D, and Kv1.2-S440A/S441A/S449A mutant channels, but Kv1.2-S440A/S441A was identical to wild-type Kv1.2 (Kv1.2-WT), and 7) bath applied 8-Br-cAMP or dialysis with PKA catalytic subunit (cPKA) increased Kv1.2-WT but not Kv1.2-S449A current amplitude. cPKA increased Kv1.2-WT current in inside-out patches. Rp-CPT-cAMPS reduced Kv1.2-WT current, blocked the increase due to 8-Br-cAMP, but had no effect on Kv1.2-S449A. cPKA increased current due to double mutant Kv1.2-S440A/S441A but had no effect on Kv1.2-S449D or Kv1.2-S440A/S441A/S449A. We conclude that Ser-449 in Kv1.2 is a site of PKA phosphorylation and a potential molecular mechanism for Kv1-containing KDR channel modulation by agonists via PKA activation.
- ItemOpen AccessMolecular basis of vascular smooth muscle potassium channels(2001) Thorneloe, Kevin; Walsh, Michael P.
- ItemOpen AccessMolecular mechanisms controlling vascular smooth muscle contractility(2008) Johnson, Rosalyn; Cole, William C.; Walsh, Michael P.
- ItemOpen AccessNovel Regulators of Vascular Myogenic Tone: A Focus on SMTNL1 and ZIPK(2018-09-19) Turner, Sara Rose; Macdonald, Justin Anthony; Walsh, Michael P.; Cole, William C.; Slater, Donna M.; Thompson, Jennifer; Nixon, Graeme F.Myogenic constriction in the resistance vasculature plays a fundamental role in the regulation of blood flow, maintenance of mean arterial pressure and in promoting overall cardiovascular health. Myogenic control of these arteries is an innate function of the vascular smooth muscle and is activated by pressure-dependent mechanisms of Ca2+-CaM-MLCK activation, Ca2+ sensitization and cytoskeletal reorganization. Recently, two proteins of interest to our research group were found to contribute to Ca2+ sensitization in vascular smooth muscle, smoothelin-like 1(SMTNL1) and zipper-interacting protein kinase (ZIPK). SMTNL1 is a relatively unknown protein which may act as an inhibitor of MLCP. ZIPK is a Ser/Thr kinase capable of phosphorylating LC20, MYPT1 and CPI-17 proteins among other targets. Neither SMTNL1 nor ZIPK has been previously investigated for a role in contributing to the regulation of the vascular myogenic response, and this investigation forms the core of this thesis. The findings presented here identify: (1) enhanced myogenic response in the mesenteric arteries of the male SMTNL1 KO mouse corresponding with (2) significant potential for Ca2+ sensitization via down regulation of MYPT1 and upregulation of CPI-17, and (3) the first evidence for ZIPK contribution to the vascular myogenic response of the rat cerebral and cremasteric arteries. While these findings were limited to the healthy vasculature, common chronic diseases such as hypertension and type 2 diabetes mellitus are known to be associated with pathological alterations in the myogenic response of the vasculature. Using a novel and specific ZIPK inhibitor, HS38, we identified a role for ZIPK activity in mediating some of the maladaptations of the vasculature in rodent models of hypertension (the SHR) and type 2 diabetes (the GK rat). In summary, these findings suggest both SMTNL1 and ZIPK play important roles in the regulation of healthy vascular function and may provide new avenues for exploration into the dysfunction of the vasculature in pathological conditions.
- ItemOpen AccessProtein biochemical and molecular biological studies of protein kinase C(1996) Andrea, Jacquelyn E.; Walsh, Michael P.
- ItemOpen AccessRegulation of cerebral arterial myogenic tone by Kv1 channels(2006) Chen, Tim; Cole, William C.; Walsh, Michael P.
- ItemOpen AccessRole of caldesmon in the regulation of smooth muscle contraction(1986) Ngai, Philip Kam-Ming; Walsh, Michael P.
- ItemOpen AccessSignal transduction via the thromboxane A2 receptor in vascular smooth muscle(2003) Susnjar, Marija; Walsh, Michael P.
- ItemOpen AccessStructural differences between various states of soybean calmodulin isoforms 1 and 4 identified by hydrogen-deuterium exchange mass spectrometry(2011) Choi, Madonna Lily Sen Yee Choi; Walsh, Michael P.; Chik, John
- ItemEmbargoThe Phosphorylation of skeletal and cardiac myofibrillar proteins by Ca2+ - and cyclic AMP-dependent protein kinases(1985) Lim, So-Young Megan, 1961-; Walsh, Michael P.
- ItemOpen AccessThe Roles of Cholesterol in Calcium Triggered Membrane Merger(2009) Churchward, Matthew Alexander; Coorssen, Jens R.; Walsh, Michael P.