RESUMO
While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made toward identifying their individual client proteins. Herein we describe the use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. A synthetic peptide library comprising 377 in vivo phosphorylation sequences from developing seed was screened using 71 recombinant A. thaliana PK. Among the initial results, we identified 23 proteins as putative clients of 17 PK. In one instance protein phosphatase inhibitor-2 (AtPPI-2) was phosphorylated at multiple-sites by three distinct PK, casein kinase1-like 10, AME3, and a Ser PK-like protein. To confirm this result, full-length recombinant AtPPI-2 was reconstituted with each of these PK. The results confirmed multiple distinct phosphorylation sites within this protein. Biochemical analyses indicate that AtPPI-2 inhibits type 1 protein phosphatase (TOPP) activity, and that the phosphorylated forms of AtPPI-2 are more potent inhibitors. Structural modeling revealed that phosphorylation of AtPPI-2 induces conformational changes that modulate TOPP binding.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Sementes/enzimologia , Motivos de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Ensaios Enzimáticos , Escherichia coli/genética , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosforilação , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Quinases/genética , Proteínas Quinases/isolamento & purificação , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/isolamento & purificação , Proteína Fosfatase 1/metabolismo , Proteínas/genética , Proteínas/isolamento & purificação , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sementes/genética , Transdução de Sinais/genéticaRESUMO
PP1 (protein phosphatase 1) is among the most conserved enzymes known, with one or more isoforms present in all sequenced eukaryotic genomes. PP1 dephosphorylates specific serine/threonine phosphoproteins as defined by associated regulatory or targeting subunits. In the present study we performed a PP1-binding screen to find putative PP1 interactors in Arabidopsis thaliana and uncovered a homologue of the ancient PP1 interactor, I-2 (inhibitor-2). Bioinformatic analysis revealed remarkable conservation of three regions of plant I-2 that play key roles in binding to PP1 and regulating its function. The sequence-related properties of plant I-2 were compared across eukaryotes, indicating a lack of I-2 in some species and the emergence points from key motifs during the evolution of this ancient regulator. Biochemical characterization of AtI-2 (Arabidopsis I-2) revealed its ability to inhibit all plant PP1 isoforms and inhibitory dependence requiring the primary interaction motif known as RVXF. Arabidopsis I-2 was shown to be a phosphoprotein in vivo that was enriched in the nucleus. TAP (tandem affinity purification)-tag experiments with plant I-2 showed in vivo association with several Arabidopsis PP1 isoforms and identified other potential I-2 binding proteins.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteína Fosfatase 1/química , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Linhagem Celular , Núcleo Celular/metabolismo , Biologia Computacional/métodos , Bases de Dados de Proteínas , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Filogenia , Epiderme Vegetal/citologia , Epiderme Vegetal/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Estruturas Vegetais/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/isolamento & purificação , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Subunidades Proteicas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The protein phosphatase 1 catalytic subunit (PP1c) and the protein phosphatase 2B (PP2B or calcineurin) catalytic subunit (CNA) contain nonconserved N-terminal regions followed by conserved phosphatase cores. To examine the role of the N-termini of these two phosphatases, we substituted the residues 1-8 of PP1c with residues 1-42 of CNA, which is designated CNA(1-42)-PP1(9-330). The activities of CNA(1-42)-PP1(9-330) were similar to those of PP2B and different from those of PP1. The chimera was at least fourfold less sensitive to inhibition by okadaic acid, but was stimulated by nickel ions and chlorogenic acid, characteristics of PP2B not of PP1. These observations suggest that the N-terminus of CNA shifts the properties of PP1 toward those of PP2B. Our findings provide evidence that the nonconserved N-terminus of PP2B not only functions as important regulatory domain but also confers itself particular characteristics. This region may be targeted for regulation of PP2B activities in vivo.
Assuntos
Calcineurina/química , Calcineurina/metabolismo , Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Calcineurina/genética , Calcineurina/isolamento & purificação , Domínio Catalítico , Ácido Clorogênico/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Vetores Genéticos , Concentração Inibidora 50 , Íons/metabolismo , Manganês/farmacologia , Dados de Sequência Molecular , Peso Molecular , Níquel/farmacologia , Ácido Okadáico/metabolismo , Ácido Okadáico/farmacologia , Proteína Fosfatase 1/análise , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/isolamento & purificação , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Spermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon. The last phase of spermatogenesis, termed spermiogenesis, involves dramatic morphological change including formation of the acrosome, elongation and condensation of the nucleus, formation of the flagella, and disposal of unnecessary cytoplasm. A prominent cytoskeletal component of the developing spermatid is the manchette, a unique microtubular structure that surrounds the nucleus of the developing spermatid and is thought to assist in both the reshaping of the nucleus and redistribution of spermatid cytoplasm. Although the molecular motor KIFC1 has been shown to associate with the manchette, its precise role in function of the manchette and the identity of its testis specific protein partners are unknown. The purpose of this study was to identify proteins in the testis that interact with KIFC1 using a yeast 2 hybrid screen of a testis cDNA library. RESULTS: Thirty percent of the interacting clones identified in our screen contain an identical cDNA encoding a 40 kD protein. This interacting protein has 4 leucine-rich repeats in its amino terminal half and is expressed primarily in the testis; therefore we have named this protein testis leucine-rich repeat protein or TLRR. TLRR was also found to associate tightly with the KIFC1 targeting domain using affinity chromatography. In addition to the leucine-rich repeats, TLRR contains a consensus-binding site for protein phosphatase-1 (PP1). Immunocytochemistry using a TLRR specific antibody demonstrates that this protein is found near the manchette of developing spermatids. CONCLUSION: We have identified a previously uncharacterized leucine-rich repeat protein that is expressed abundantly in the testis and associates with the manchette of developing spermatids, possibly through its interaction with the KIFC1 molecular motor. TLRR is homologous to a class of regulatory subunits for PP1, a central phosphatase in the reversible phosphorylation of proteins that is key to modulation of many intracellular processes. TLRR may serve to target this important signaling molecule near the nucleus of developing spermatids in order to control the cellular rearrangements of spermiogenesis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Proteína Fosfatase 1/genética , Proteínas/genética , Espermátides/metabolismo , Espermátides/fisiologia , Espermatogênese/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Diferenciação Celular/genética , Proteínas do Citoesqueleto , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Repetições Ricas em Leucina , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Especificidade de Órgãos , Ligação Proteica , Proteína Fosfatase 1/química , Proteína Fosfatase 1/isolamento & purificação , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas/isolamento & purificação , Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Testículo/metabolismo , beta Carioferinas/metabolismoRESUMO
BACKGROUND: Protein phosphatase one (PP1) is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. RESULTS: We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIalpha, several nuclear helicases, NUP153 and the TRRAP complex. CONCLUSION: This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.
Assuntos
Cromatografia de Afinidade/métodos , Proteína Fosfatase 1/isolamento & purificação , Motivos de Aminoácidos , Animais , Sítios de Ligação , Glicogênio/isolamento & purificação , Células HeLa , Humanos , Microcistinas/química , Microcistinas/metabolismo , Músculo Esquelético/metabolismo , Proteína Fosfatase 1/química , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Coelhos , Sefarose/químicaRESUMO
We developed a quantitative method to measure the activity of cyclin-dependent kinases (Cdks) by western blotting, without radioisotopes. We prepared a recombinant protein substrate based upon the natural Cdk1 substrate, PP1Cα. By combining this substrate in a western blot method using fluorochrome based antibodies and phospho-imager analysis, we measured the Km of ATP binding to Cdk1 to be 3.5 µM. We then measured Cdk1 activity in cell extracts from interphase or mitotic cells, and demonstrated that previously identified Cdk inhibitors could be detected by this assay. Our data show that we have a safe, reliable assay to identify Cdk1 inhibitors and measure Cdk1 activity.
Assuntos
Western Blotting/métodos , Proteína Quinase CDC2/análise , Proteína Fosfatase 1/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequência de Aminoácidos , Western Blotting/normas , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Escherichia coli/genética , Expressão Gênica , Células HT29 , Humanos , Células MCF-7 , Mitose/genética , Dados de Sequência Molecular , Fosforilação , Proteína Fosfatase 1/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Type-1 phosphatase (PP-1) was assessed in foot muscle (FM) and hepatopancreas (HP) of estivating (EST) Otala lactea. Snail PP-1 displayed several conserved traits, including sensitivity to inhibitors, substrate affinity, and reduction in size to a 39 kDa catalytic subunit (PP-1c). During EST, PP-1 activity in FM and HP crude extracts was reduced, though kinetics and protein levels of purified PP-1c isoforms were not altered. PP-1c protein levels increased and decreased in nuclear and glycogen-associated fractions, respectively, during EST. Gel filtration determined that a 257 kDa low K(m) PP-1alpha complex decreased during estivation whereas a 76 kDa high K(m) complex increased in EST. Western blotting confirmed that the 76 kDa protein consisted of PP-1alpha and nuclear inhibitor of PP-1 (NIPP-1). A suppression of PP-1 activity factors in the overall metabolic rate depression in estivating snails and the mechanism is mediated through altered cellular localization and interaction with binding partners.