Activated regulatory and memory T-cells accumulate in malignant ascites from ovarian carcinoma patients.

Invasive ovarian cancer is associated with poor outcome. The presence of infiltrating regulatory T-cells (Tregs) suppresses protective anti-tumor immune responses, and their accumulation into the tumor microenvironment correlates with reduced survival in ovarian cancer patients. Here, we conducted a detailed characterization of CD4(+) T-cells, CD8(+) T-cells and Treg subsets in the peripheral blood and malignant ascites fluid from seventeen patients with ovarian carcinoma of epithelial origin. Cell distribution, activation status and proliferation status were assessed by multi-color flow cytometry. In ascites fluid, a significant accumulation of CD8(+) cytotoxic T-cells and Tregs was observed compared to peripheral blood. Furthermore, a skewing toward the CD45RA(-) effector/memory compartment was observed in all T-cell subsets in the ascites fluid, but was most pronounced in the Treg population. Regulatory T-cells in the malignant ascites were more activated and had a higher proliferation rate compared to blood-derived cells from the same patient, and their number in ascites was positively correlated with the number of epithelial cells in effusion. In summary, we demonstrate an accumulation of activated CD4(+), CD8(+) and regulatory T-cells in the cancer microenvironment of ovarian carcinoma.

ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequence requirements for LC3-interacting region (LIR) motifs.

Autophagy is a lysosome-dependent degradation system conserved among eukaryotes. The mammalian Atg1 homologues, Unc-51 like kinase (ULK) 1 and 2, are multifunctional proteins with roles in autophagy, neurite outgrowth, and vesicle transport. The mammalian ULK complex involved in autophagy consists of ULK1, ULK2, ATG13, FIP200, and ATG101. We have used pulldown and peptide array overlay assays to study interactions between the ULK complex and six different ATG8 family proteins. Strikingly, in addition to ULK1 and ULK2, ATG13 and FIP200 interacted with human ATG8 proteins, all with strong preference for the GABARAP subfamily. Similarly, yeast and Drosophila Atg1 interacted with their respective Atg8 proteins, demonstrating the evolutionary conservation of the interaction. Use of peptide arrays allowed precise mapping of the functional LIR motifs, and two-dimensional scans of the ULK1 and ATG13 LIR motifs revealed which substitutions that were tolerated. This information, combined with an analysis of known LIR motifs, provides us with a clearer picture of sequence requirements for LIR motifs. In addition to the known requirements of the aromatic and hydrophobic residues of the core motif, we found the interactions to depend strongly on acidic residues surrounding the central core LIR motifs. A preference for either a hydrophobic residue or an acidic residue following the aromatic residue in the LIR motif is also evident. Importantly, the LIR motif is required for starvation-induced association of ULK1 with autophagosomes. Our data suggest that ATG8 proteins act as scaffolds for assembly of the ULK complex at the phagophore.

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells.

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.

T cell-signaling network analysis reveals distinct differences between CD28 and CD2 costimulation responses in various subsets and in the MAPK pathway between resting and activated regulatory T cells.

To uncover signaling system differences between T cell stimuli and T cell subsets, phosphorylation status of 18 signaling proteins at six different time points following TCR triggering and CD28/CD2 costimulation was examined in human T cell subsets by phospho-epitope-specific flow cytometry of fluorescent cell barcoded samples, thereby providing a high-resolution signaling map. Compared with effector/memory T cells, naive T cells displayed stronger activation of proximal signaling molecules after TCR triggering alone. Conversely, distal phosphorylation events, like pErk and pS6-ribosomal protein, were stronger in effector/memory subsets. CD28 costimulation specifically induced signaling necessary for proper NF-κB activation, whereas CD2 signaled more strongly to S6-ribosomal protein. Analysis of resting regulatory T cells (rTregs; CD4(+)CD45RA(+)FOXP3(+)) and activated regulatory T cells (actTregs; CD4(+)CD45RA(-)FOXP3(++)) revealed that, although rTregs had low basal, but inducible, Erk activity, actTregs displayed high basal Erk phosphorylation and little or no Akt activation. Interestingly, the use of Mek inhibitors to block Erk activation inhibited activation-dependent FOXP3 upregulation in rTregs, their transition to actTregs, and the resulting increase in suppressive capacity. In summary, our systems approach unraveled distinct differences in signaling elicited by CD28 and CD2 costimulation and between rTregs and actTregs. Blocking rTreg transition to highly suppressive actTregs by Mek inhibitors might have future therapeutic applications.

PI3K p110delta regulates T-cell cytokine production during primary and secondary immune responses in mice and humans.

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110delta during the activation and differentiation of naive T cells, and p110delta inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110delta activity is required for interferon-gamma production. Moreover, acute inhibition of p110delta inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110delta played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor-induced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110delta inhibition as naive T cells and show that mouse models accurately predict p110delta function in human T cells. There is therefore a strong rationale for p110delta inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases.

Quantitative proteome analysis of detergent-resistant membranes identifies the differential regulation of protein kinase C isoforms in apoptotic T cells.

Several lines of evidence suggest that detergent-resistant membranes (DRMs) (also known as lipid rafts and glycosphingolipid-enriched microdomains) may have a role in signaling pathways of apoptosis. Here, we developed a method that combines DRMs isolation and methanol/chloroform extraction with stable isotope labeling with amino acids in cell culture-based quantitative proteome analysis of DRMs from control and cisplatin-induced apoptotic Jurkat T cells. This approach enabled us to enrich proteins with a pivotal role in cell signaling of which several were found with increased or decreased amounts in DRMs upon induction of apoptosis. Specifically, we show that three isoforms of protein kinase C (PKC) are regulated differently upon apoptosis. Although PKC alpha which belongs to the group of conventional PKCs is highly up-regulated in DRMs, the levels of two novel PKCs, PKC eta and PKC theta, are significantly reduced. These alterations/differences in PKC regulation are verified by immunoblotting and confocal microscopy. In addition, a specific enrichment of PKC alpha in apoptotic blebs and buds is shown. Furthermore, we observe an increased expression of ecto-PKC alpha as a result of exposure to cisplatin using flow cytometry. Our results demonstrate that in-depth proteomic analysis of DRMs provides a tool to study differential localization and regulation of signaling molecules important in health and disease.

Splicing factor arginine/serine-rich 17A (SFRS17A) is an A-kinase anchoring protein that targets protein kinase A to splicing factor compartments.

Protein kinase A (PKA) is targeted to distinct subcellular localizations by specific protein kinase A anchoring proteins (AKAPs). AKAPs are divided into subclasses based on their ability to bind type I or type II PKA or both. Dual-specificity AKAPs were recently reported to have an additional PKA binding determinant called the RI specifier region. A bioinformatic search with the consensus RI specifier region identified a novel AKAP, the splicing factor arginine/serine-rich 17A (SFRS17A). Here, we show by a variety of protein interaction assays that SFRS17A binds both type I and type II PKA in vitro and inside cells, demonstrating that SFRS17A is a dual-specific AKAP. Moreover, immunofluorescence experiments show that SFRS17A colocalizes with the catalytic subunit of PKA as well as the splicing factor SC35 in splicing factor compartments. Using the E1A minigene splicing assay, we found that expression of wild type SFRS17A conferred regulation of E1A alternative splicing, whereas the mutant SFRS17A, which is unable to bind PKA, did not. Our data suggest that SFRS17A is an AKAP involved in regulation of pre-mRNA splicing possibly by docking a pool of PKA in splicing factor compartments.

Systematic review of sequencing of ALK inhibitors in ALK-positive non-small-cell lung cancer.

The objective of this study was to understand outcomes of patients treated with ALK inhibitors, especially when ALK inhibitors are followed by other ALK inhibitors. A systematic literature review was conducted in PubMed, Embase, and Cochrane through July 17, 2017. Conference abstracts (three meetings in past 2 years) also were searched. Of 504 unique publications, 80 met inclusion criteria (47 clinical trials, 33 observational studies). Observational studies have the potential to provide information for ALK inhibitors used sequentially. Ten observational studies reported median overall survival of crizotinib-led sequences ranging from 30.3 to 63.75 months from initiation of crizotinib; 49.4-89.6 months from metastatic non-small-cell lung cancer diagnosis; and 15.5-22.0 months from initiation of the second-generation ALK inhibitor after initial crizotinib. Sequencing of ALK inhibitors may benefit patients progressing on initial ALK inhibitors.

CD8+ regulatory T cells-A distinct T-cell lineage or a transient T-cell phenotype?

Regulation of the immune system is fundamental for proper immune function and homeostasis. In the periphery, regulatory T cells provide self-tolerance and protect the host from harmful autoreactive T cells. Regulatory T cells are also involved in the pathogenesis of chronic viral infectious diseases and cancer. Both the CD4(+) and the CD8(+) T cell compartments contain regulatory T cells. Although various CD4(+) regulatory T-cell subsets represent the best characterized regulatory T-cell populations, CD8(+) regulatory T cells also have strong immunosuppressive properties and are involved in the pathogenesis of various clinical conditions. In this review, we will discuss the phenotypic and functional characteristics of CD8(+) regulatory T cells in comparison to CD4(+) regulatory T cells.

Overall survival in renal cell carcinoma after introduction of targeted therapies: a Norwegian population-based study.

BACKGROUND: This population-wide retrospective, non-interventional registry study assessed changes in overall survival (OS) and factors influencing OS in Norwegian patients with renal cell carcinoma (RCC). METHODS: Two population-wide health registries were used to identify all RCC patients with (mRCC) or without metastases diagnosed before (2002-2005) and after (2006-2008 and 2009-2011) introduction of targeted therapies. Median OS was estimated using Kaplan-Meier method. Cox proportional hazards regression modeling was used to identify prognostic factors. RESULTS: Overall, 5,463 patients were diagnosed with RCC during 2002-2005 (n=1,898), 2006-2008 (n=1,631), and 2009-2011 (n=1,934); of these, 1,678 (31%) had mRCC. Patients diagnosed in 2009-2011 and 2006-2008 had significant (P<0.001) improvements in OS versus those diagnosed in 2002-2005: median OS, not reached and not reached versus 82.0 months in RCC; 14.0 and 12.0 months versus 9.0 months in mRCC. Similarly, OS improvements were seen in the primary and elderly (≥75 years) mRCC populations. Median OS was comparable (12 months) between clear cell and papillary mRCC, but it was longer (24.0 months) for chromophobe mRCC. Multivariate regression analyses showed that younger age, previous nephrectomy, and 1 or more prescriptions of targeted therapy were significantly associated with longer OS in mRCC patients. CONCLUSION: OS increased in RCC and mRCC patients in Norway between 2002 and 2011 following introduction of targeted therapies.

Molecular mechanisms for protein kinase A-mediated modulation of immune function.

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.

Cyclic AMP inhibits translation of cyclin D3 in T lymphocytes at the level of elongation by inducing eEF2-phosphorylation.

The purpose of the present study was to understand the mechanism by which activated protein kinase A (PKA) leads to down-regulation of cyclin D3 in lymphocytes. By using Jurkat cells as a model system, we have been able to demonstrate that cyclin D3 is reduced at the level of translation by inhibition of elongation. One of the important factors involved in translational elongation is the eukaryotic elongation factor 2 (eEF2). eEF2 promotes translation in its unphosphorylated form, and we observed a rapid phosphorylation of the eEF2-protein upon forskolin treatment. When using specific inhibitors of the eEF2-kinase prior to forskolin treatment, we were able to inhibit the increased phosphorylation of eEF2. Furthermore, inhibition of eEF2-kinase prevented the forskolin-mediated down-regulation of cyclin D3. Taken together, it appears that activation of PKA in Jurkat cells reduces the expression of cyclin D3 at the level of translational elongation by increasing the phosphorylation of eEF2 and thereby inhibiting its activity.

Docking of PRAK/MK5 to the atypical MAPKs ERK3 and ERK4 defines a novel MAPK interaction motif.

ERK3 and ERK4 are atypical MAPKs in which the canonical TXY motif within the activation loop of the classical MAPKs is replaced by SEG. Both ERK3 and ERK4 bind, translocate, and activate the MAPK-activated protein kinase (MK) 5. The classical MAPKs ERK1/2 and p38 interact with downstream MKs (RSK1-3 and MK2-3, respectively) through conserved clusters of acidic amino acids, which constitute the common docking (CD) domain. In contrast to the classical MAPKs, the interaction between ERK3/4 and MK5 is strictly dependent on phosphorylation of the SEG motif of these kinases. Here we report that the conserved CD domain is dispensable for the interaction of ERK3 and ERK4 with MK5. Using peptide overlay assays, we have defined a novel MK5 interaction motif (FRIEDE) within both ERK4 and ERK3 that is essential for binding to the C-terminal region of MK5. This motif is located within the L16 extension lying C-terminal to the CD domain in ERK3 and ERK4 and a single isoleucine to lysine substitution in FRIEDE totally abrogates binding, activation, and translocation of MK5 by both ERK3 and ERK4. These findings are the first to demonstrate binding of a physiological substrate via this region of the L16 loop in a MAPK. Furthermore, the link between activation loop phosphorylation and accessibility of the FRIEDE interaction motif suggests a switch mechanism for these atypical MAPKs in which the phosphorylation status of the activation loop regulates the ability of both ERK3 and ERK4 to bind to a downstream effector.

Reduced Cbl phosphorylation and degradation of the zeta-chain of the T-cell receptor/CD3 complex in T cells with low Lck levels.

T cells with short interfering RNA-mediated Lck-knockdown (kd) display paradoxical hyper-responsiveness upon TCR ligation. We have previously reported a possible mechanism for T-cell activation in cells with low levels of Lck depending on Grb2-SOS1 recruitment to the zeta-chain of TCR/CD3 (Methi et al., Eur. J. Immunol. 2007, 37: 2539-2548). Here, we show that short interfering RNA-mediated targeting of Lck caused a dramatic reduction in c-Cbl phosphorylation and a general reduction in protein ubiquitination after TCR stimulation. Specifically, this resulted in reduced ubiquitination of the zeta-chain, yet internalization of TCR/CD3 appeared to be normal after receptor engagement. However, zeta-chain levels were elevated in Lck-kd cells, and confocal microscopy revealed reduced colocalization of CD3-containing vesicles with endosomal and lysosomal compartments. We hypothesize that prolonged stability of internalized T-cell receptor complex may result in extended signaling in T cells with low Lck levels.

Regulation of FynT function by dual domain docking on PAG/Cbp.

In resting T-cells, the transmembrane adaptor protein PAG (phosphoprotein associated with glycosphingolipid-enriched microdomains) is constitutively tyrosine-phosphorylated, a state maintained by the Src family kinase FynT. PAG has a role in negative regulation of Src family kinases in T-cells by recruitment of Csk (C-terminal Src kinase) to the membrane via binding to PAG phosphotyrosine 317. The interaction between FynT and PAG is essential for PAG function; however, so far the FynT binding mode has been unknown. Here, we demonstrate that the FynT-PAG complex formation is a dual domain docking process, involving SH2 domain binding to PAG phosphotyrosines as well as an SH3 domain interaction with the first proline-rich region of PAG. This binding mode affects FynT kinase activity, PAG phosphorylation, and recruitment of FynT and Csk, demonstrated in Jurkat TAg cells after antibody stimulation of the T cell receptor. Furthermore, we show that TCR-induced tyrosine phosphorylation is regulated by SH3 domain modulation of the FynT-PAG interaction in human primary T-cells. Although FynT SH3 domain association is shown to be crucial for efficiently initiating PAG phosphorylation, we suggest that engagement of the SH2 domain on PAG renders FynT insensitive to Csk negative regulation. Thus, in T-cells, PAG is involved in positive as well as negative regulation of FynT activity.

Generation of highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) regulatory T cells by continuous antigen stimulation.

Continuous antigen stimulation of CD4(+)CD25(-) T cells leads to generation of adaptive CD4(+)CD25(+)FOXP3(+) regulatory T (T(R)) cells. Here, we show that highly suppressive adaptive CD8(+)CD25(+)FOXP3(+) T cells can be generated in the same manner by continuous antigen stimulation in the presence of CD14(+) monocytes. During the course of stimulation, acquisition of immunosuppressive properties develops in parallel with up-regulation and expression of cytotoxic molecules. The CD8(+) T(R) cells inhibit CD4(+) and CD8(+) T cell proliferation and cytokine production, but do not alter the expression of granzyme A and granzyme B or perforin in CD8(+) effector T cells. Although, the CD8(+) T(R) cells express prostaglandin E(2), IL-10 and TGF-beta, the mechanism of suppression was independent of these soluble factors. In contrast to adaptive CD4(+) T(R) cells, the CD8(+) T(R) cells suppress mainly by a contact-dependent mechanism as evident from transwell experiments. However, neither blocking antibodies to CTLA-4, CD80 nor CD86 could reverse CD8(+) T(R)-mediated suppression, indicating that other mechanism(s) must be employed by these cells.

The heterotrimeric G-protein alpha-subunit Galphaq regulates TCR-mediated immune responses through an Lck-dependent pathway.

Here, we examined the functional involvement of heterotrimeric G-proteins in TCR-induced immune responses. TCR/CD3 crosslinking resulted in activation of both Galphaq and Galphas, but not Galphai-2. Targeting of Galphas, Galphai-2 and Galphaq using siRNA demonstrated a specific role of Galphaq in TCR signaling. Jurkat TAg T cells with Galphaq knockdown displayed reduced activation of Lck and LAT phosphorylation, but paradoxically showed sustained ERK1/2 phosphorylation and increased NFAT-AP-1-reporter activity implicating Galphaq in the negative control of downstream signaling and IL-2-promoter activity. Primary T cells isolated from Galphaq-deficient mice had a similar TCR signaling response with reduced proximal LAT phosphorylation, sustained ERK1/2 phosphorylation and augmented immune responses including increased secretion of IL-2, IL-5, IL-12 and TNF-alpha. The effects on NFAT-AP-1-reporter activity were sensitive to the Src family kinase inhibitor PP2 and were reversed by transient expression of constitutively active Lck. Furthermore, expression of constitutively active Galphaq Q209L elevated Lck activity and Zap-70 phosphorylation. Together these data argue for a role of Galphaq in the fine-tuning of proximal TCR signals at the level of Lck and a negative regulatory role of Galphaq in transcriptional activation of cytokine responses.

The rat orthologue to the inhibitory receptor gp49B is expressed by neutrophils and monocytes, but not by NK cells or mast cells.

Mouse gp49B is a member of the leukocyte immunoglobulin-like receptor family. It is constitutively expressed by mast cells and certain myeloid cells, and expression can be induced on natural killer (NK) cells and T cells. We have cloned several rat cDNA, 78% identical to mouse gp49B at the amino acid level, that represent the rat orthologue to mouse gp49B. A mouse monoclonal antibody (WEN29) against rat gp49B was generated. By flow cytometry and Northern blot analysis, gp49B was found to be expressed by neutrophils and monocytes, but not NK cells (primary or IL-2-activated), T cells (resting or concanavalin A-stimulated) or peritoneal mast cells. Following pervanadate treatment, the tyrosine phosphatase SHP-1 was co-immunoprecipitated with gp49B in the macrophage cell line R2. In glutathione S-transferase pull-down experiments, the cytoplasmic tail of rat gp49B associated with the SH2 domains of both SHP-1 and SHP-2, dependent on intact and phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM). Compared to mouse, the cytoplasmic domain of rat gp49B contains a third ITIM-like sequence (YLYASV) that was phosphorylated by several Src family tyrosine kinases, enhanced the phosphorylation of other ITIM, and bound to the SH2 domains of SHP-2, suggesting a role in the recruitment of downstream phosphatases.

Lck is required for stromal cell-derived factor 1 alpha (CXCL12)-induced lymphoid cell chemotaxis.

Stromal cell-derived factor 1alpha (CXCL12) induces chemotaxis of lymphocytes through its receptor CXCR4. We examined the role of nonreceptor tyrosine kinases in CXCL12-induced chemotaxis of T cells and natural killer (NK) cells. Damnacanthal, a specific Lck inhibitor, but not the Syk inhibitor piceatannol, inhibited CXCL12-induced chemotaxis of both lymphocyte subsets. Similarly, damnacanthal was shown to inhibit CXCL12-induced chemotaxis of the Jurkat T-cell line. Stimulating T and NK cells with CXCL12 increased both the tyrosine phosphorylation and the kinase activity of Lck. A direct involvement of Lck in CXCL12-induced chemotaxis was demonstrated in the Lck-deficient Jurkat-derived cell line JCaM1.6. Although JCaM1.6 cells express CXCR4, no significant migration was detected after CXCL12 stimulation. Reconstitution with wild-type Lck restored both CXCL12-induced chemotaxis and Lck activation. Furthermore, cotransfection of wild-type Lck with C-terminal Src kinase (Csk) into JCaM1.6 failed to restore the chemotactic response induced by CXCL12. Finally, by targeting critical residues in the Src homology-2 (SH2) or SH3 domains of Lck, we observed that the SH3 domain is important for the function of Lck in CXCL12-mediated chemotaxis. Together, these results suggest a role for Lck in CXCL12-induced signaling pathways leading to lymphocyte chemotaxis.

Activation of C-terminal Src kinase (Csk) by phosphorylation at serine-364 depends on the Csk-Src homology 3 domain.

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.