RESUMO
The down-regulation or cellular depletion of protein kinase C (PKC) attendant to prolonged activation by phorbol esters is a widely described property of this key family of signaling enzymes. However, neither the mechanism of down-regulation nor whether this mechanism occurs following stimulation by physiological agonists is known. Here we show that the peptidyl-prolyl isomerase Pin1 provides a timer for the lifetime of conventional PKC isozymes, converting the enzymes into a species that can be dephosphorylated and ubiquitinated following activation induced by either phorbol esters or natural agonists. The regulation by Pin1 requires both the catalytic activity of the isomerase and the presence of a Pro immediately following the phosphorylated Thr of the turn motif phosphorylation site, one of two C-terminal sites that is phosphorylated during the maturation of PKC isozymes. Furthermore, the second C-terminal phosphorylation site, the hydrophobic motif, docks Pin1 to PKC. Our data are consistent with a model in which Pin1 binds the hydrophobic motif of conventional PKC isozymes to catalyze the isomerization of the phospho-Thr-Pro peptide bond at the turn motif, thus converting these PKC isozymes into species that can be efficiently down-regulated following activation.
Assuntos
Peptidilprolil Isomerase/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/metabolismo , Proteína Quinase C/metabolismo , Sequência de Aminoácidos , Animais , Bombesina/farmacologia , Células COS , Chlorocebus aethiops , Regulação para Baixo/fisiologia , Fibroblastos/citologia , Proteínas de Choque Térmico HSP70/metabolismo , Células HeLa , Humanos , Isoenzimas/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Peptidilprolil Isomerase de Interação com NIMA , Neurotransmissores/farmacologia , Peptidilprolil Isomerase/genética , Proteína Quinase C beta , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ratos , Especificidade por Substrato/fisiologia , Ubiquitinação/fisiologiaRESUMO
Beclin 1, a subunit of the class III phosphatidylinositol 3-kinase complex, is a tumour suppressor with a central role in endocytic trafficking, cytokinesis and the cross-regulation between autophagy and apoptosis. Interestingly, not only reduced expression but also overexpression of Beclin 1 is correlated with cancer development and metastasis. Thus it seems necessary for the cell to balance the protein levels of Beclin 1. In the present study we describe a regulatory link between Beclin 1 and the ubiquitin ligase Nedd4 (neural-precursor-cell-expressed developmentally down-regulated 4). We establish Nedd4 as a novel binding partner of Beclin 1 and demonstrate that Nedd4 polyubiquitinates Beclin 1 with Lys11- and Lys63-linked chains. Importantly, Nedd4 expression controls the stability of Beclin 1, and depletion of the Beclin 1-interacting protein VPS34 causes Nedd4-mediated proteasomal degradation of Beclin 1 via Lys11-linked polyubiquitin chains. Beclin 1 is thus the first tumour suppressor reported to be controlled by Lys11-linked polyubiquitination.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteína Beclina-1 , Classe III de Fosfatidilinositol 3-Quinases/genética , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Ubiquitina-Proteína Ligases Nedd4 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Estabilidade Proteica , Proteólise , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoAssuntos
Anticorpos/química , Artefatos , Indicadores e Reagentes/química , Proteínas Nucleares/análise , Fosfoproteínas Fosfatases/análise , beta Catenina/análise , Anticorpos/imunologia , Células CACO-2 , Reações Cruzadas/imunologia , Reações Falso-Positivas , Expressão Gênica , Humanos , Imuno-Histoquímica , Indicadores e Reagentes/normas , Espectrometria de Massas , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Proto-Oncogene Mas , beta Catenina/genéticaRESUMO
Protein kinase A (PKA) is a serine/threonine kinase that regulates a number of cellular processes important for immune activation and control. Modulation of signal transduction by PKA is a complex and diverse process, and differential isozyme expression, holoenzyme composition and subcellular localization contribute specificity to the PKA signalling pathway. In lymphocytes, phosphorylation by PKA has been demonstrated to regulate antigen receptor-induced signalling both by altering protein-protein interactions and by changing the enzymatic activity of target proteins. PKA substrates involved in immune activation include transcription factors, members of the MAP kinase pathway and phospholipases. The ability of PKA type I to regulate activation of signalling components important for formation of the immunological synapse, demonstrates that the cAMP signalling pathway can directly modulate proximal events in lymphocyte activation. Furthermore, the recent discovery that PKA regulates Src kinases through modulation of Csk, supports the notion that PKA is involved in the fine-tuning of immune receptor signalling in lipid rafts.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Linfócitos T/imunologia , Proteína Tirosina Quinase CSK , Imunodeficiência de Variável Comum/enzimologia , Imunodeficiência de Variável Comum/imunologia , Infecções por HIV/enzimologia , Infecções por HIV/imunologia , Humanos , Ativação Linfocitária , Microdomínios da Membrana/enzimologia , Modelos Biológicos , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/enzimologia , Quinases da Família srcRESUMO
The class III phosphatidylinositol 3-kinase (PI3K-III) complex and its phosphorylated lipid product phosphatidylinositol 3-phosphate (PtdIns3P) control the three topologically related membrane-involution processes autophagy, endocytosis, and cytokinesis. The activity of the catalytic unit of PI3K-III complex, the Vacuolar sorting protein 34 (VPS34), depends on the membrane targeting unit Vacuolar sorting protein 15 (VPS15), and the tumor suppressor protein Beclin 1. It is established that the overall activity of VPS34 is positively regulated by Beclin 1, whose positive influence is further controlled through the association with a set of Beclin1 interacting components, which stimulate or inhibit VPS34. The interaction between Beclin 1 and Beclin 1-associated components are controllable and is regulated by phosphorylation in a context-dependent manner. Here, we focus on an emerging concept whereby the activity of the PI3K-III complex is controlled by ubiquitination of Beclin 1 or Beclin 1-associated molecules. In summary, at least three different ubiquitin ligases can affect the positive regulatory function of Beclin 1 towards VPS34, suggesting that ubiquitination is an important and physiologically relevant event in tuning the tumor suppressor function of Beclin 1.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Genes Supressores de Tumor , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Fosfatidilinositol 3-Quinase/metabolismo , Ubiquitinação , Animais , Proteína Beclina-1 , Humanos , Neoplasias/enzimologia , FosforilaçãoRESUMO
Certain secreted proteins bypass the canonical exit pathway from cells. Two studies now shed light on the unconventional secretion route taken by the yeast acyl-coenzyme A-binding protein: this protein is sequestered into autophagic vesicles that are re-routed to the plasma membrane where their content is released to the extracellular space.
Assuntos
Inibidor da Ligação a Diazepam/metabolismo , Via Secretória/fisiologia , Vesículas Secretórias/fisiologia , Autofagia/fisiologia , Proteínas de Transporte/metabolismo , Membrana Celular/fisiologia , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Macroautophagy is a cellular process whereby the cell sequesters and recycles cytosolic constituents in a lysosome-dependent manner. It has also been implicated in a number of disorders, including cancer and neurodegeneration. Although a previous report that AGS3 over-expression promotes macroautophagy suggests a stimulatory role of AGS3 in this process, we have found that knock-down of AGS3, unexpectedly, also induces macroautophagy, indicating an inhibitory function of endogenous AGS3 in macroautophagy. Interestingly, AGS3 phosphorylation is decreased upon induction of mammalian target of rapamycin (mTOR)-dependent macroautophagy. Moreover, unlike wild-type AGS3, over-expression of an AGS3 mutant lacking this modification fails to enhance macroautophagic activity. These observations imply that AGS3 phosphorylation may participate in the modulation of macroautophagy.
Assuntos
Autofagia/fisiologia , Proteínas de Transporte/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Western Blotting , Proteínas de Transporte/genética , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Técnicas de Inativação de Genes , Inibidores de Dissociação do Nucleotídeo Guanina , Humanos , Imunoprecipitação , Mutagênese Sítio-Dirigida , Fosforilação , Reação em Cadeia da Polimerase , Serina-Treonina Quinases TORRESUMO
Engagement of the T-cell receptor (TCR) in human primary T cells activates a cyclic AMP (cAMP)-protein kinase A (PKA)-Csk inhibitory pathway that prevents full T-cell activation in the absence of a coreceptor stimulus. Here, we demonstrate that stimulation of CD28 leads to recruitment to lipid rafts of a beta-arrestin/phosphodiesterase 4 (PDE4) complex that serves to degrade cAMP locally. Redistribution of the complex from the cytosol depends on Lck and phosphatidylinositol 3-kinase (PI3K) activity. Protein kinase B (PKB) interacts directly with beta-arrestin to form part of the supramolecular complex together with sequestered PDE4. Translocation is mediated by the PKB plextrin homology (PH) domain, thus revealing a new role for PKB as an adaptor coupling PI3K and cAMP signaling. Functionally, PI3K activation and phosphatidylinositol-(3,4,5)-triphosphate (PIP3) production, leading to recruitment of the supramolecular PKB/beta-arrestin/PDE4 complex to the membrane via the PKB PH domain, results in degradation of the TCR-induced cAMP pool located in lipid rafts, thereby allowing full T-cell activation to proceed.
Assuntos
Arrestinas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Arrestinas/genética , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Ativação Enzimática , Humanos , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Alinhamento de Sequência , Linfócitos T/metabolismo , beta-ArrestinasRESUMO
cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.
Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteína Quinase Tipo I Dependente de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Proteínas do Citoesqueleto/fisiologia , Imunossupressores/farmacologia , Microdomínios da Membrana/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas de Ancoragem à Quinase A/química , Proteínas de Ancoragem à Quinase A/metabolismo , Actinas/metabolismo , Células Cultivadas , Clonagem Molecular , Proteína Quinase Tipo I Dependente de AMP Cíclico/antagonistas & inibidores , Proteína Quinase Tipo I Dependente de AMP Cíclico/genética , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Humanos , Células Jurkat , Microdomínios da Membrana/metabolismo , Mapeamento de Peptídeos , Fosfoproteínas/fisiologia , Ligação Proteica , RNA Interferente Pequeno/farmacologia , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Trocadores de Sódio-Hidrogênio/fisiologia , Linfócitos T/enzimologiaRESUMO
Regulation of Src kinase activity is tightly coupled to the phosphorylation status of the C-terminal regulatory tyrosine Tyr(527), which, when phosphorylated by Csk, represses Src. Here, we demonstrate that activation of Csk through a prostaglandin E(2)-cAMP-protein kinase A (PKA) pathway inhibits Src. This inhibitory pathway is operative in detergent-resistant membrane fractions where cAMP-elevating agents activate Csk, resulting in a concomitant decrease in Src activity. The inhibitory effect on Src depends on a detergent-resistant membrane-anchored Csk and co-localization of all components of the inhibitory pathway in membrane microdomains. Furthermore, epidermal growth factor-induced activation of Src and phosphorylation of the Src substrates Cbl and focal adhesion kinase are inhibited by activation of the cAMP-PKA-Csk pathway. We propose a novel mechanism whereby G protein-coupled receptors inhibit Src signaling by activation of Csk in a cAMP-PKA-dependent manner.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Proteína Tirosina Quinase CSK , AMP Cíclico/metabolismo , Humanos , Células Jurkat , Fosforilação , Domínios de Homologia de src , Quinases da Família srcRESUMO
Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E(2) (PGE(2)) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE(2) reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3 zeta-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE(2)-mediated inhibition of TCR-induced zeta-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase A-mediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR.
Assuntos
AMP Cíclico/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Complexo CD3/metabolismo , Proteína Tirosina Quinase CSK , Divisão Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Humanos , Interleucina-2/genética , Microdomínios da Membrana/metabolismo , Peptídeos/química , Fosforilação , Fosfotransferases/metabolismo , Testes de Precipitina , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismo , Tirosina/metabolismo , Domínios de Homologia de src , Quinases da Família srcRESUMO
Signal transducer and activator of transcription (STAT) proteins may be activated by epidermal growth factor (EGF), but their role in EGF receptor-mediated mitogenic signaling is not clear. We previously showed that Stat5b was activated by EGF in rat hepatocytes in primary monolayer culture. In the present study, we found that EGF induced tyrosine phosphorylation of Stat5b both on Tyr-699, which correlated with specific DNA binding activity, and also on other tyrosine residues. The Src tyrosine kinase inhibitor CGP77675 blocked the EGF-induced activation of Stat5b, but did not affect the Stat5b activation by growth hormone (GH) or prolactin (PRL). The Stat5b response to EGF was most pronounced soon (3 h) after plating (early G(1)) and at high cell density (50,000 hepatocytes per cm(2)). However, at this cell density EGF did not stimulate DNA synthesis. In hepatocytes at 24 h of culturing (mid/late G(1)) with 20,000 cells per cm(2), EGF induced strong phosphorylation of the EGF receptor, as well as Shc and ERK, and stimulated DNA synthesis, but did not activate Stat5b, although the Stat5b response to GH or PRL was retained. A strong GH-induced Stat5b activation neither influenced the DNA synthesis alone nor enhanced the mitogenic effect of EGF. The results show that EGF induces tyrosine phosphorylation and DNA-binding activity of Stat5b in a manner different from GH and PRL, apparently by a Src-dependent mechanism. The data also provide further evidence that Stat5b is not required for mitogenic signaling from the EGF receptor.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Receptores ErbB/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Proteínas do Leite , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transativadores/metabolismo , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , DNA/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Regulação para Baixo/efeitos dos fármacos , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/farmacologia , Hormônio do Crescimento/farmacologia , Hepatócitos/efeitos dos fármacos , Masculino , Mitógenos/farmacologia , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Prolactina/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Pirimidinas/farmacologia , Pirróis/farmacologia , Ratos , Ratos Wistar , Fator de Transcrição STAT5 , Transativadores/antagonistas & inibidores , Fator de Crescimento Transformador alfa/farmacologiaRESUMO
C-terminal Src kinase (Csk) controls the Src family kinase Lck, which is essential for T cell antigen receptor (TCR)-mediated signaling. For the first time, we here report the effects of acute elimination of Csk in Jurkat T cells and primary T cells using short interfering (si) RNA. In both cell types, 70-85% knockdown of Csk was achieved within 48 h. No alterations in surface expression of CD3, CD4 or CD8, or in Lck protein level were observed. Phosphorylation of Y505 in Lck was markedly reduced and a concomitant 4-5-fold increase in Lck Y394 phosphorylation was observed both in normal and Jurkat T cells. Kinase assays revealed 2-3-fold higher Lck activity. In Jurkat cells, basal levels of zeta chain phosphorylation were elevated, and spontaneous NFAT-AP-1 activation occurred, indicating aberrant Lck kinase activity. After TCR triggering, Csk knockdown cells revealed faster and stronger, but not sustained, phosphorylation of Lck Y394 and zeta chains compared to control. TCR-induced activation of NFAT-AP-1 and TCR/CD28-stimulated IL-2 secretion occurred at weaker stimuli and with augmented responses in Csk knockdown Jurkat and primary T cells, respectively. Altogether, these data suggest that acute elimination of Csk in T cells without evolution of compensatory mechanisms results in aberrant Lck activity and augmented TCR-stimulated responses.
Assuntos
Proteínas Tirosina Quinases/genética , Interferência de RNA/fisiologia , RNA Interferente Pequeno/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Proteína Tirosina Quinase CSK , Humanos , Células Jurkat , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/enzimologia , Quinases da Família srcRESUMO
Ligation of the TCR along with the coreceptor CD28 is necessary to elicit T cell activation in vivo, whereas TCR triggering alone does not allow a full T cell response. Upon T cell activation of human peripheral blood T cells, we found that the majority of cAMP was generated in T cell lipid rafts followed by activation of protein kinase A. However, upon TCR and CD28 coligation, beta-arrestin in complex with cAMP-specific phosphodiesterase 4 (PDE4) was recruited to lipid rafts which down-regulated cAMP levels. Whereas inhibition of protein kinase A increased TCR-induced immune responses, inhibition of PDE4 blunted T cell cytokine production. Conversely, overexpression of either PDE4 or beta-arrestin augmented TCR/CD28-stimulated cytokine production. We show here for the first time that the T cell immune response is potentiated by TCR/CD28-mediated recruitment of PDE4 to lipid rafts, which counteracts the local, TCR-induced production of cAMP. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a coreceptor stimulus.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/fisiologia , Antígenos CD28/fisiologia , Microdomínios da Membrana/enzimologia , Receptores de Antígenos de Linfócitos T/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Arrestinas/metabolismo , AMP Cíclico/biossíntese , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Humanos , Ativação Linfocitária , Transdução de Sinais , Linfócitos T/imunologia , beta-ArrestinasRESUMO
In the present study, we investigate the mechanism for the protein kinase A (PKA)-mediated activation of C-terminal Src kinase (Csk). Although isolated Csk kinase domain was phosphorylated at Ser(364) by PKA to the same stoichiometry as wild-type Csk, significant activation of the isolated Csk kinase domain by PKA was observed only in the presence of the purified Src homology 3 domain (SH3 domain). Furthermore, the interaction between the SH3 and kinase domains was facilitated by PKA-mediated phosphorylation of the kinase domain, as evaluated by surface plasmon resonance. This suggests that an overall structural domain organization and interaction between the kinase and SH3 domains are important for the activity of Csk and its regulation by PKA.