Molecular Mechanism of Zipper Interacting Protein Kinase
Diseases caused by smooth muscle dysfunction such as hypertension and asthma are major public health concerns, a better understanding of the signaling pathways that regulate smooth muscle contraction could identify new drug targets. The opposing effects of two enzymes; calcium/calmodulin regulated myosin light chain kinase (MLCK) and smooth muscle myosin phosphatase (SMPP-1M) determine the amount of force generated by smooth muscle. The calcium-independent signaling mediated by myosin phosphatase is regulated by several kinases which include zipper interacting protein kinase (ZIPK). Our laboratory has shown that ZIPK is able to phosphorylate and inhibit SMPP-1M which results in increased smooth muscle contraction. Additional studies demonstrated that ZIPK is also regulated by phosphorylation. The goal of this study is to identify kinases in the context of smooth muscle that regulate ZIPK and to define the events required for ZIPK activation.
A proteomic approach which employed ATP-affinity chromatography coupled with mass spectrometry isolated discreet kinase activities towards ZIPK, these activities were attributed to integrin-linked kinase (ILK) and Rho kinase 1 (ROCK1). ILK phosphorylates ZIPK at Thr180 while ROCK1 phosphorylates ZIPK at Thr265 and Thr299.
Additionally the ATP-affinity media used for kinase enrichment in the proteomic screen was used as a tool to measure ZIPK activation. Pre-incubating ZIPK with ROCK before the assay resulted in increased binding which suggests phosphorylation of ZIPK by ROCK is activating. Increasing the substrate concentration in the assay resulted in increased ZIPK binding, this result was only observed when the assay was performed with the full-length protein. Phosphorylation of residues in the kinase domain along with substrate binding relieves inhibition and results in kinase activation.
Finally fluorescence microscopy along with targeted mutations of ZIPK was used to determine the mechanism of cellular transport. This was done to address the difference in cellular localization between human and murine cells. The localization of human ZIPK is dictated by nuclear localization sequence 2 (NLS2) and the phosphorylation state of Thr299; the mechanism is not shared by the murine form of ZIPK.
Completion of this work has provided additional information about the signaling interactions that take place in smooth muscle; the results suggest that ZIPK is a convergence point for multiple signaling pathways that lead to SMPP-1 inhibition and subsequently smooth muscle contraction. This study also contributes significantly to our knowledge of the molecular dynamics that lead to active full length ZIPK. Future research that employs animal modeling as a tool to investigate ZIPK will be informed by the experiments that address the cellular localization of ZIPK.
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