RESUMO
The polyomavirus middle T antigen (PyMT) oncogene activates the cellular nonreceptor tyrosine kinase c-Src and recruits the Hippo pathway effectors, Yap (yes-associated protein) and Taz (transcriptional coactivator with PDZ-binding motif), as key steps in oncogenesis. Yap and Taz are transcription coactivators shuttling from the cytoplasm to the nucleus. The Hippo pathway kinase Lats1/2 (large tumor suppressor homolog) reduces Yap/Taz nuclear localization and minimizes their cytoplasmic levels by facilitating their ubiquitination by the E3 ligase SCF(ß-TrCP). In contrast, PyMT increases the cytoplasmic Taz level. Here we show that this unique PyMT behavior is mediated by Src. We demonstrate that PyMT-induced Src activation inhibits degradation of both wild-type and tyrosine-less Taz, ruling out Taz modification as a mechanism of escaping degradation. Instead, we found that Src attenuates the SCF(ß-TrCP) E3-ligase activity in blunting Taz proteasomal degradation. The role of Src in rescuing Taz from TrCP-mediated degradation gives rise to higher cell proliferation under dense cell culture. Finally, IkB (NF-kappa-B inhibitor), a known substrate of ß-TrCP, was rescued by Src, suggesting a wider effect of Src on ß-TrCP substrates. These findings introduce the Src tyrosine kinase as a regulator of SCF(ß-TrCP).
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Contendo Repetições de beta-Transducina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/metabolismo , Proteína Tirosina Quinase CSK , Linhagem Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Células HCT116 , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Células NIH 3T3 , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Sinalização YAP , Proteínas Contendo Repetições de beta-Transducina/genética , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Adipocyte differentiation, or adipogenesis, is a complex and highly regulated process. A recent proteomic analysis has predicted that the nonreceptor tyrosine kinase Abelson murine leukemia viral oncogene (c-Abl) is a putative key regulator of adipogenesis, but the underlying mechanism remained obscure. We found that c-Abl was activated during the early phase of mouse 3T3-L1 preadipocyte differentiation. Moreover, c-Abl activity was essential and its inhibition blocked differentiation to mature adipocytes. c-Abl directly controlled the expression and activity of the master adipogenic regulator peroxisome proliferator-activator receptor gamma 2 (PPARγ2). PPARγ2 physically associated with c-Abl and underwent phosphorylation on two tyrosine residues within its regulatory activation function 1 (AF1) domain. We demonstrated that this process positively regulates PPARγ2 stability and adipogenesis. Remarkably, c-Abl binding to PPARγ2 required the Pro12 residue that has a phenotypically well-studied common human genetic proline 12 alanine substitution (Pro12Ala) polymorphism. Our findings establish a critical role for c-Abl in adipocyte differentiation and explain the behavior of the known Pro12Ala polymorphism.
Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , PPAR gama/fisiologia , Proteínas Proto-Oncogênicas c-abl/fisiologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Animais , Benzamidas/farmacologia , Células HEK293 , Humanos , Mesilato de Imatinib , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , PPAR gama/química , PPAR gama/genética , Fosforilação , Fosfotirosina/química , Piperazinas/farmacologia , Mutação Puntual , Polimorfismo de Nucleotídeo Único , Prolina/química , Ligação Proteica , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Inibidores de Proteínas Quinases/farmacologia , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/química , Pirimidinas/farmacologia , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transcrição GênicaRESUMO
The Hippo pathway utilizes a well-characterized Ser/Thr kinase cascade to control the downstream effectors, Yap and Taz. In addition, Yap/Taz and other Hippo pathway components are directly regulated by tyrosine kinases (TKs). The methodological strategies described here use the example of the c-Abl non-receptor TK and the Yap substrate to outline the steps used to identify and to validate tyrosine phosphorylation sites, including bioinformatic approaches, ectopic expression of proteins in transfected tissue culture cells, and mutagenesis of endogenous proteins by CRISPR-Cas9. These general strategies can be applied to investigate regulation of protein signaling moieties by tyrosine phosphorylation in the context of distinct TKs.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Biomarcadores , Linhagem Celular , Biologia Computacional/métodos , Via de Sinalização Hippo , Humanos , Mutação , Fosforilação , Análise de Sequência de DNA , Software , Fatores de Transcrição/metabolismo , NavegadorRESUMO
A number of key regulatory proteins contain one or two copies of the WW domain known to mediate protein-protein interaction via proline-rich motifs, such as PPxY. The Hippo pathway components take advantage of this module to transduce tumor suppressor signaling. It is becoming evident that tyrosine phosphorylation is a critical regulator of the WW proteins. Here, we review the current knowledge on the involved tyrosine kinases and their roles in regulating the WW proteins.