Abstract
Excitation-contraction coupling in smooth muscle involves activation of myosin light chain (MLC) phosphorylation, which increases activity of the myosin actin-activated ATPase, resulting in contraction. Phosphorylation of MLC phosphatase (SMPP-1M) by Rho-associated kinase or endogenous SMPP-1M-associated kinase inhibits SMPP-1M, enhancing MLC phosphorylation and contraction. However, the precise identity of SMPP-1M-associated kinase remains unclear. Biochemical evidence strongly supports the idea that SMPP-1M-associated kinase is related to the human serine/threonine leucine zipper-interacting protein kinase (hZIPK), which is important in cell apoptosis, and the SMPP-1M-associated kinase has therefore been called ZIP-like kinase (MacDonald, J. A., Borman, M. A., Murani, A., Somlyo, A. V., Hartshorne, D. J., and Haystead, T. A. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2419-2424). Whether the vascular smooth muscle SMPP-1M-associated kinase is a truncated version of hZIPK, native hZIPK, or a unique homologue of hZIPK is unclear. Here we show that only native hZIPK mRNA and protein are detectable in human vascular smooth muscle cells (VSMCs). High stringency screening of a human aortic cDNA library for the SMPP-1M-associated kinase identified 18 positive clones, all of which proved to be clones of hZIPK. PCR-based studies of VSMC RNA revealed native hZIPK transcripts but no evidence for splice variants of hZIPK or a ZIP-like kinase. Northern blotting studies of multiple vascular and non-vascular tissue RNAs, including human bladder RNA, showed only 2.3 kb of mRNA predicted for full-length hZIPK. Immunoblotting showed native full-length 52-kDa hZIPK expression in VSMCs. Full-length and N-terminal hZIPK bound the C-terminal domain (amino acids 681-847) of the myosin binding subunit (MBS) of SMPP-1M. hZIPK immunoprecipitated with the MBS of SMPP-1M and dominant negative RhoA inhibited the hZIPK-MBS interaction. These data identify hZIPK as the unique SMPP-1-associated kinase expressed in human vesicular smooth muscle and support a role for Rho in promoting the hZIPK-MBS interaction.
Highlights
Blood vessel tone regulates blood pressure and flow and is itself dynamically regulated by the contractile state of vascular smooth muscle cells (VSMCs)1 in the blood vessel wall
We show that only native human serine/ threonine leucine zipper-interacting protein kinase (hZIPK) mRNA and protein are detectable in human vascular smooth muscle cells (VSMCs)
Contraction and relaxation of VSMCs is determined by the phosphorylation state of myosin light chains (MLCs), a process that is tightly regulated by the opposing activities of myosin light chain kinase and myosin phosphatase (SMPP-1M) [1, 2]
Summary
Blood vessel tone regulates blood pressure and flow and is itself dynamically regulated by the contractile state of vascular smooth muscle cells (VSMCs) in the blood vessel wall. Contraction and relaxation of VSMCs is determined by the phosphorylation state of myosin light chains (MLCs), a process that is tightly regulated by the opposing activities of myosin light chain kinase and myosin phosphatase (SMPP-1M) [1, 2]. RhoA, when activated by vasoconstrictors, binds and activates its effector Rho-kinase, which leads to SMPP-1M phosphorylation [13]. As recent data support the physiologic importance of SMPP-1M regulation, attention has focused on identification of the SMPP-1Massociated kinase(s). ZIP-like kinase was isolated from bladder smooth muscle as a 32-Da phosphoprotein that co-purified with SMPP-1M, phosphorylated SMPP-1M at inhibitory residues, and was activated by the smooth muscle contractile agonist carbachol [16]. Despite data supporting a physiological role for a ZIP-like kinase in smooth muscle, the precise identity of this kinase and
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
