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1.
Cell Signal ; 23(1): 297-303, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20854903

RESUMO

ZIPK (zipper-interacting protein kinase) is a Ca(2+)-independent protein kinase that promotes myosin phosphorylation in both smooth muscle and non-muscle cells. A recent report attempted to clarify a debate over the subcellular localization of ZIPK in non-muscle cells (Shoval et. al. (2007) Plos Genetics. 3: 1884-1883). A species-specific loss of a key phosphorylation site (T299) in murine (mouse and rat) ZIPK seems to direct it to the nucleus, while the presence of the T299 site in human ZIPK correlates with cytoplasmic localization. T299 is immediately adjacent to a putative nuclear localization sequence (NLS) and may mask its function when phosphorylated, therefore explaining the species-specific dichotomy of intracellular localization. However, despite the murine ZIPK (mZIPK) lacking the T299 residue that is critical for controlling human ZIPK (hZIPK) subcellular localization, mutational analysis showed that this NLS control locus is nonfunctional in the murine context. A constitutively active Rho promoted the cytoplasmic retention of a human ZIPK mutant that would otherwise localize to the nucleus. Endogenous hZIPK showed sensitivity to the nuclear export inhibitor leptomycin B, suggesting a continuous shuttling between cytoplasm and nucleus that is dependent upon T299 dephosphorylation. Thus, the C-terminal domain of human and murine ZIPK demonstrated quite divergent nuclear import and export functionality. We conclude that in the case of ZIPK, studies between the species may not be directly comparable to each other given the gross differences in intracellular localization and movement.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Núcleo Celular/enzimologia , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/análise , Proteínas Reguladoras de Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/análise , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Associadas com Morte Celular , Ácidos Graxos Insaturados/farmacologia , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Mutação , Fosforilação , Especificidade da Espécie , Proteínas rho de Ligação ao GTP/metabolismo
2.
J Biol Chem ; 282(7): 4884-4893, 2007 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-17158456

RESUMO

Zipper-interacting protein kinase (ZIPK) regulates Ca(2+)-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.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Músculo Liso/enzimologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Proteínas Quinases Dependentes de Cálcio-Calmodulina , Morte Celular/fisiologia , Proteínas Quinases Associadas com Morte Celular , Ativação Enzimática/fisiologia , Masculino , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Miosinas/metabolismo , Peptídeos/farmacologia , Fosforilação , Proteômica , Fibras de Estresse/metabolismo , Suínos , Quinases Associadas a rho
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