Hiroshi Hosoya

ROCK1 Phosphorylates and Activates Zipper-interacting Protein Kinase

Zipper-interacting protein kinase (ZIPK) regulates Ca2+-independent phosphorylation of both smooth muscle (to regulate contraction) and non-muscle myosin (to regulate non-apoptotic cell death) through either phosphorylation and inhibition of myosin phosphatase, the myosin phosphatase inhibitor CPI17, or direct phosphorylation of myosin light chain. ZIPK is regulated by multisite phosphorylation. Phosphorylation at least three sites Thr-180, Thr-225, and Thr-265 has been shown to be essential for full activity, whereas phosphorylation at Thr-299 regulates its intracellular localization. Herein we utilized an unbiased proteomics screen of smooth muscle extracts with synthetic peptides derived from the sequence of the regulatory phosphorylation sites of the enzyme to identify the protein kinases that might regulate ZIPK activity in vivo. Discrete kinase activities toward Thr-265 and Thr-299 were defined and identified by mass spectrometry as Rho kinase 1 (ROCK1). In vitro, ROCK1 showed a high degree of substrate specificity toward native ZIPK, both stoichiometrically phosphorylating the enzyme at Thr-265 and Thr-299 as well as bringing about activation. In HeLa cells, coexpression of ZIPK with ROCK1 altered the ROCK-induced phenotype of focused stress fiber pattern to a Rho-like phenotype of parallel stress fiber pattern. This effect was also dependent upon phosphorylation at Thr-265. Our findings provide a new regulatory pathway in smooth muscle and non-muscle cells whereby ROCK1 phosphorylates and regulates ZIP kinase.

Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase.

Death-associated protein kinase (DAP kinase) is a proapoptotic, calcium/calmodulin-dependent serine/threonine kinase. Here, we report that DAP kinase phosphorylates the regulatory light chain of myosin II (MLC) both in vitro and in vivo, and that this phosphorylation occurs preferentially at residue Ser19. In quiescent fibroblasts, DAP kinase stabilizes stress fibers through phosphorylation of MLC, but it is dispensable for the formation of peripheral microfilament bundles. This cytoskeletal effect of DAP kinase occurs before the onset of apoptosis and does not require an intact death domain. In addition, DAP kinase is required for serum-induced stress-fiber formation, which is associated with the upregulation of its catalytic activity. Despite being both sufficient and necessary for the assembly or maintenance of stress fibers, DAP kinase is incapable of stimulating the formation of focal adhesions in quiescent cells. Moreover, it promotes the disassembly of focal adhesions but not stress fibers in cells receiving serum factors. Together, our results identify a novel and unique function of DAP kinase in the uncoupling of stress fibers and focal adhesions. Such uncoupling would lead to a perturbation of the balance between contractile and adhesion forces and subsequent cell detachment, which might contribute to its pro-apoptotic activity.

The carboxy-terminal pleckstrin homology domain of ROCK interacts with filamin-A.

The small GTPase Rho and its effector ROCK/Rho-kinase regulate actin cytoskeletal reorganization through phosphorylation of the regulatory light chain of myosin II. We previously reported that ROCK co-purified with the actin-binding protein filamin-A from HeLa cells. Here, we show that the pleckstrin homology (PH) domain of ROCK, but not the kinase or coiled-coil domain, interacts with filamin-A. We also determined that the PH domain of ROCK binds to the carboxy-terminal region of filamin-A containing the last 24th repeat. ROCK co-localized with filamin-A at the protrusive cell membranes of HeLa cells.

Myosin II Regulatory Light Chain Is Required for Trafficking of Bile Salt Export Protein to the Apical Membrane in Madin-Darby Canine Kidney Cells

BSEP, MDR1, and MDR2 ATP binding cassette transporters are targeted to the apical (canalicular) membrane of hepatocytes, where they mediate ATP-dependent secretion of bile acids, drugs, and phospholipids, respectively. Sorting to the apical membrane is essential for transporter function; however, little is known regarding cellular proteins that bind ATP binding cassette proteins and regulate their trafficking. A yeast two-hybrid screen of a rat liver cDNA library identified the myosin II regulatory light chain, MLC2, as a binding partner for BSEP, MDR1, and MDR2. The interactions were confirmed by glutathione S-transferase pulldown and co-immunoprecipitation assays. BSEP and MLC2 were overrepresented in a rat liver subcellular fraction enriched in canalicular membrane vesicles, and MLC2 colocalized with BSEP in the apical domain of hepatocytes and polarized WifB, HepG2, and Madin-Darby canine kidney cells. Expression of a dominant negative, non-phosphorylatable MLC2 mutant reduced steady state BSEP levels in the apical domain of polarized Madin-Darby canine kidney cells. Pulse-chase studies revealed that Blebbistatin, a specific myosin II inhibitor, severely impaired delivery of newly synthesized BSEP to the apical surface. These findings indicate that myosin II is required for BSEP trafficking to the apical membrane.

Phosphorylation of myosin II regulatory light chain is necessary for migration of HeLa cells but not for localization of myosin II at the leading edge

To investigate the role of phosphorylated myosin II regulatory light chain (MRLC) in living cell migration, these mutant MRLCs were engineered and introduced into HeLa cells. The mutant MRLCs include an unphosphorylatable form, in which both Thr-18 and Ser-19 were substituted with Ala (AA-MRLC), and pseudophosphorylated forms, in which Thr-18 and Ser-19 were replaced with Ala and Asp, respectively (AD-MRLC), and both Thr-18 and Ser-19 were replaced with Asp (DD-MRLC). Mutant MRLC-expressing cell monolayers were mechanically stimulated by scratching, and the cells were forced to migrate in a given direction. In this wound-healing assay, the AA-MRLC-expressing cells migrated much more slowly than the wild-type MRLC-expressing cells. In the case of DD-MRLC- and AD-MRLC-expressing cells, no significant differences compared with wild-type MRLC-expressing cells were observed in their migration speed. Indirect immunofluorescence staining showed that the accumulation of endogenous diphosphorylated MRLC at the leading edge was not observed in AA-MRLC-expressing cells, although AA-MRLC was incorporated into myosin heavy chain and localized at the leading edge. In conclusion, we propose that the phosphorylation of MRLC is required to generate the driving force in the migration of the cells but not necessary for localization of myosin II at the leading edge.