MacDonald, JustinAl-Ghabkari, Abdulhameed2017-05-302017-05-3020172017Al-Ghabkari, A. (2017). Regulation of Vascular Smooth Muscle Cell Motility by Protein Kinases: A Focus on Zipper-Interacting Protein Kinase (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/27591http://hdl.handle.net/11023/3844Zipper-interacting protein kinase (ZIPK) is a protein serine/threonine kinase that mediates a variety of cellular functions. ZIPK is a key regulator of vascular smooth muscle (VSM) cell contractility and motility. Further investigations for the upstream regulators, activation signals, downstream target(s), regulatory mechanism(s) and the role of ZIPK in different signaling modules are required. Two major strategies were utilized in this study to examine ZIPK signaling in VSM cells; chemical genetics and application of small molecule inhibitors. For the chemical genetics strategy, ZIPK was engineered in the ATP-binding pocket by replacing a bulky amino acid (Leu) with a small one (Gly). This ZIPK-L93G kinase was introduced into VSM cells and then the NM-PPI inhibitor (selective for L93G-ZIPK, not for WT-ZIPK) was applied, which demonstrated an inhibitory effect on myosin phosphorylation. This strategy provides another approach to study ZIPK signaling, especially in the absence of specific inhibitors for ZIPK. Recent investigations led to the development of two small molecule inhibitors for ZIPK; DI and HS-38. The selectivity of the DI compound was assessed by; (i) in vitro studies; (ii) in situ analysis; and (iii) in silico molecular modelling. Our studies revealed that the DI compound was not selective for ZIPK; DI had effects on the cellular cytoskeleton and motility that were associated with diminution of ROCKII signaling pathways. The HS-38 compound was advantageous to delineate a novel signaling relationship between ZIPK and focal adhesion kinase (FAK). ZIPK inhibition by HS-38 or ZIPK knockdown with siRNA resulted in suppression of pY397-FAK phosphorylation and remodeling of the cytoskeletal architecture. Additional molecular details of this signaling mechanism implicate a member of the dual specificity phosphatase family (CDC14A) in a putative partnership with ZIPK to regulate focal adhesion dynamics and FAK phosphorylation.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.BiochemistryProtein kinasesVascular smooth muscle motilitySmall molecule inhibitorsdoctoral thesis10.11575/PRISM/27591