RESUMO
Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues 46-60) and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Fosfatase 2/química , Proteína Fosfatase 2/metabolismo , Ubiquitinação/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/genética , Espectroscopia de Ressonância Magnética , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Modelos Biológicos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , Ubiquitinação/genéticaRESUMO
With the recent clinical success of drugs targeting protein kinase activity, drug discovery efforts are focusing on the role of reversible protein phosphorylation in disease states. The activity of protein phosphatases, enzymes that oppose protein kinases, can also be manipulated to alter cellular signaling for therapeutic benefits. In this review, we present protein serine/threonine phosphatases as viable therapeutic targets, discussing past successes, current challenges, and future strategies for modulating phosphatase activity.
Assuntos
Descoberta de Drogas , Fosfoproteínas Fosfatases/fisiologia , Proteínas Tirosina Fosfatases/fisiologia , Animais , Antineoplásicos/farmacologia , Domínio Catalítico , Ativação Enzimática , Humanos , Imunossupressores/farmacologia , Complexos Multiproteicos/antagonistas & inibidores , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosforilação , Proteínas Tirosina Fosfatases/antagonistas & inibidoresRESUMO
Protein serine/threonine phosphatase (PP2A) is a major cellular enzyme implicated in the control of numerous signaling processes. The accurate measurement of PP2A activity in crude cell lysates, immune complexes, and purified preparations provides insight into the function and regulation of this essential enzyme, which, in turn, can lead to a better understanding of the signaling pathways that it modulates. The method presented here utilizes 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) and a FLEXstation for the continuous measure of PP2A activity associated with many different protein preparations. This automated fluorescence-based assay offers several distinct advantages over colorimetric and radioactive assays of phosphatase activity including (1) decreased substrate preparation time, (2) real-time kinetic data, (3) high sensitivity, and (4) the capability to analyze a wide variety of phosphatases.
Assuntos
Fluorescência , Monoéster Fosfórico Hidrolases/metabolismo , Himecromona/análogos & derivados , Himecromona/química , Biologia Molecular/métodos , Monoéster Fosfórico Hidrolases/química , Reprodutibilidade dos TestesRESUMO
The protein serine/threonine phosphatase (PP) type 2A family consists of three members: PP2A, PP4, and PP6. Specific rabbit and sheep antibodies corresponding to each catalytic subunit, as well as a rabbit antibody recognizing all three subunits, were utilized to examine the expression of these enzymes in select rat tissue extracts. PP2A, PP4, and PP6 catalytic subunits (PP2A(C), PP4(C), and PP6(C), respectively) were detected in all rat tissue extracts examined and exhibited some differences in their levels of expression. The expression of alpha4, an interacting protein for PP2A family members that may function downstream of the target of rapamycin (Tor), was also examined using specific alpha4 sheep antibodies. Like the phosphatase catalytic subunits, alpha4 was ubiquitously expressed with particularly high levels in the brain and thymus. All three PP2A family members, but not alpha4, bound to the phosphatase affinity resin microcystin-Sepharose. The phosphatase catalytic subunits were purified to apparent homogeneity (PP2A(C) and PP4(C)) or near homogeneity (PP6(C)) from bovine testes soluble extracts following ethanol precipitation and protein extraction. In contrast to PP2A(C), PP4(C) and PP6(C) exhibited relatively low phosphatase activity towards several substrates. Purified PP2A(C) and native PP2A in cellular extracts bound to GST-alpha4, and co-immunoprecipitated with endogenous alpha4 and ectopically expressed myc-tagged alpha4. The interaction of PP2A(C) with alpha4 was unaffected by rapamycin treatment of mammalian cells; however, protein serine/threonine phosphatase inhibitors such as okadaic acid and microcystin-LR disrupted the alpha4/PP2A complex. Together, these findings increase our understanding of the biochemistry of alpha4/phosphatase complexes and suggest that the alpha4 binding site within PP2A may include the phosphatase catalytic domain.
