A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper-interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998)
Dlk/ZIP kinase is a serine/threonine kinase highly homologous to DAP kinase. We have reported that HeLa ZIP kinase (hZIPK) phosphorylated the regulatory light chain of myosin II (MRLC) at both Ser19 and Thr18 in vitro. In this study, we demonstrate that hZIPK also induces the diphosphorylation of MRLC in nonmuscle cells. Peptide mapping revealed that transient transfection of hZIPK into HeLa cells caused diphosphorylation of MRLC. In contrast, transfection of the kinase inactive mutant of hZIPK did not induce any phosphorylation of MRLC. Using antibodies speci®c for mono-or diphosphorylated MRLC, we showed that diphosphorylated MRLC induced by the overexpression of hZIPK was concentrated in striking aggregates or bundles of actin ®laments in HeLa cells, while monophosphorylated MRLC showed no prominent localization to these aggregates. Overexpression of hZIPK also induced dramatic changes in cell shape and disruption of nuclear morphology reminiscent of changes during apoptosis. These e ects of hZIPK were suppressed by the coexpression of a mutant MRLC where both phosphorylation sites were replaced with alanine, indicating that the changes in actin organization were a consequence of MRLC diphosphorylation. These results suggested that hZIPK plays a role in regulating actin organization and cell morphology in non-muscles and at least part of its e ects are mediated through the diphosphorylation of MRLC. Oncogene (2001) 20, 8175 ± 8183.
ABSTRACT. Activity of nonmuscle myosin II is regulated by phosphorylation of its regulatory light chain (MRLC). Phosphoryration of MRLC at both Thr18 and Ser19 (diphosphorylation) results in higher MgATPase activity and in promotion of the assembly of myosin II filaments than does that of MRLC at Ser19 (monophosphorylation) in vitro. To determine the roles of the diphosphorylated MRLC in vivo, we transfected three kinds of MRLC mutants, unphosphorylated, monophosphorylated and diphosphorylated forms (MRLC2 T18AS19A , substitution of both Ser19 and Thr18 by Ala; MRLC2 T18AS19D , Ser19 by Asp and Thr18 by Ala; and MRLC2 T18DS19D , both Ser19 and Thr18 by Asp, respectively), into HeLa cells. Cells overexpressing the mutant MRLC2 T18DS19D contained a larger number of actin filament bundles than did those overexpressing the mutant MRLC2 T18AS19D . Moreover, cells overexpressing the nonphosphorylatable mutant MRLC2 T18AS19A showed a decrease in the number of actin filament bundles. Taken together, our data suggest that diphosphorylation of MRLC plays an important role in regulating actin filament assembly and reorganization in nonmuscle cells. Key words: phosphorylation/myosin II filament/myosin II regulatory light chain/cytokinesisMyosin II is an essential protein to generate force for cell locomotion (Berlot et al., 1985(Berlot et al., , 1987 and cytokinesis (Mabuchi and Okuno, 1977;De Lozanne and Spudich, 1987). In smooth muscle and vertebrate nonmuscle cells, myosin II activity is both negatively and positively regulated by phosphorylation of its regulatory light chain (MRLC). The phosphorylation sites of MRLC by Ca 2+ /calmodulindependent myosin light chain kinase (MLCK) are mainly Ser19 (monophosphorylation) and, under some conditions, both Ser19 and Thr18 (diphosphorylation) in vitro (Ikebe and Hartshorne, 1985;Ikebe et al., 1986). Phosphorylation of MRLC by MLCK enhances the actin activated Mg ATPase activity (Sellers et al., 1981;Trybus, 1989) and promotes the assembly of myosin II filaments (Suzuki et al., 1978). These effects are more effective when MRLC is diphosphorylated (Ikebe et al., 1988). On the other hand, protein kinase C (PKC) phosphorylates MRLC at Ser1/2 and Thr9. Phosphorylation of MRLC by PKC down-regulate the MgATPase activity of myosin II phosphorylated by MLCK (Nishikawa et al., 1984;Ikebe et al., 1987).It has been reported that substitution of amino acid residues on phosphorylation sites of MRLC affects the biochemical properties of myosin II in vitro. Substitution of both Ser19 and Thr18 to Asp, which mimics the diphosphorylated MRLC, exerted the actin activated MgATPase activity of myosin II and formation of myosin II filaments (Kamisoyama et al., 1994;Sweeney et al., 1994).In this study, we attempted to determine the roles of diphosphorylated MRLC in vivo. To address this, we transfected three kinds of MRLC mutants, which mimic nonphosphorylated, monophosphorylated and diphosphorylated MRLC, into HeLa cells and compared their localization in both interphase and mitotic cells. Materials ...
Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)‐containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide‐ and 5‐hydroxythalidomide‐induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell‐free protein synthesis system, PLZF was identified as a thalidomide‐dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5‐hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5‐hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide‐dependent CRBN substrate SALL4, was induced by thalidomide or 5‐hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide‐induced phenotypes. Our findings implicate PLZF as an important thalidomide‐induced CRBN neosubstrate involved in thalidomide teratogenicity.
Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis.
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