Src Kinase-associated Phosphoprotein Research Articles

SKAP2 acts downstream of CD11b/CD18 and regulates neutrophil effector function.

The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involvedinthe induction of the high-affinity binding CD11b/CD18 conformation,thesignaling pathways that orchestrate this response remain incompletely understood. We performed an unbiased screening method for protein selection by biotin identification (BioID) and investigated the KINDLIN3 interactome. We used liquid chromatography with tandem mass spectrometry as a powerful analytical tool. Generation of NB4 CD18, KINDLIN3, or SKAP2 knockout neutrophils was achieved using CRISPR-Cas9 technology, and the cells were examined for their effector function using flow cytometry, live cell imaging, microscopy, adhesion, or antibody-dependent cellular cytotoxicity (ADCC). Among the 325 proteins significantly enriched, we identified Src kinase-associated phosphoprotein 2 (SKAP2), a protein involved in actin polymerization and integrin-mediated outside-in signaling. CD18 immunoprecipitation in primary or NB4 neutrophils demonstrated the presence of SKAP2 in the CD11b/CD18 complex at a steady state. Under this condition, adhesion to plastic, ICAM-1, or fibronectin was observed in the absence of SKAP2, which could be abrogated by blocking the actin rearrangements with latrunculin B. Upon stimulation of NB4 SKAP2-deficient neutrophils, adhesion to fibronectin was enhanced whereas CD18 clustering was strongly reduced. This response corresponded with significantly impaired CD11b/CD18-dependent NADPH oxidase activity, phagocytosis, and cytotoxicity against tumor cells. Our results suggest that SKAP2 has a dual role. It may restrict CD11b/CD18-mediated adhesion only under resting conditions, but its major contribution lies in the regulation of dynamic CD11b/CD18-mediated actin rearrangements and clustering as required for cellular effector functions of human neutrophils.

SKAP1 Is a Novel Biomarker and Therapeutic Target for Gastric Cancer: Evidence from Expression, Functional, and Bioinformatic Analyses.

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide. Due to the lack of early symptoms, GC is often diagnosed at an advanced stage when treatment options are limited. There is an urgent need to identify biomarkers for early detection, prognosis evaluation, and targeted treatment of GC. Studies have shown that Src kinase-associated phosphoprotein 1 (SKAP1) promotes cell proliferation and invasion and is associated with poor prognosis in colorectal cancer, malignant fibrous histiocytoma, and breast cancer. However, the role and mechanism of SKAP1 in GC are unclear. Here, analyses of multiple databases and experiments revealed that SKAP1 expression was higher in GC than in adjacent normal tissues. The Cancer Genome Atlas data showed that high SKAP1 expression was associated with poor GC prognosis. SKAP1 expression was higher in GC than in normal gastric epithelial cells. SKAP1 silencing reduced the proliferation, migration and invasion of the GC cell lines MKN45 and HGC27. Rescue experiments suggest that SKAP1 may promote GC progression by activating JAK1/PI3K/AKT signaling and regulating GC cell proliferation, invasion, migration, and other functions. Bioinformatics analysis revealed that SKAP1 was associated with immune cell infiltration and checkpoint expression in GC. High SKAP1 expression was associated with poorer immunotherapy outcomes, suggesting its potential as a predictive biomarker of GC immunotherapy efficacy. In summary, SKAP1 is overexpressed in GC, where it promotes cell proliferation, invasion and migration and is associated with poor prognosis and poor immunotherapy outcomes. SKAP1 may represent a biomarker and therapeutic target in GC and regulates cellular functions through JAK1/PI3K/AKT signaling.

Multi-functional adaptor SKAP1: regulator of integrin activation, the stop-signal, and the proliferation of T cells.

T-cell activation is a complex process involving a network of kinases and downstream molecular scaffolds or adaptors that integrate surface signals with effector functions. One key immune-specific adaptor is Src kinase-associated phosphoprotein 1 (SKAP1), which is also known as src kinase-associated protein of 55 kDa (SKAP55). This mini-review explains how SKAP1 plays multiple roles in regulating integrin activation, the "stop-signal", and the optimization of the cell cycling of proliferating T cells through interactions with various mediators, including the Polo-like kinase 1 (PLK1). Ongoing research on SKAP1 and its binding partners will likely provide important insights into the regulation of immune function and have implications for the development of new treatments for disease states such as cancer and autoimmunity.

SKAP2 is downregulated in the villous tissues of patients with missed abortion and regulates growth and migration in trophoblasts through the WAVE2-ARP2/3 signaling pathway

IntroductionAbnormal placental trophoblast function is the main cause of missed abortion (MA). Src kinase-associated phosphoprotein 2 (SKAP2) indirectly affects actin reunion, which is significantly associated with cell migration. MethodsTwenty women with MA and 20 healthy women who underwent voluntarily induced abortion were included in this study. Immunohistochemistry, qRT-PCR, and western blotting were used to determine SKAP2, WAVE2, and ARP2 expression in the villous tissues. We investigated the effects of SKAP2 and the W336K mutant (blocked SKAP2 Src homology 3 function) on growth and migration in HTR8/SVneo cells using the CCK8 assay, flow cytometry, and transwell assay. The effects of SKAP2 on the WAVE2-ARP2/3 signaling pathway in HTR8/SVneo cells were evaluated by western blotting and immunofluorescence. ResultsCompared to the women in the voluntary abortion group, SKAP2 and WAVE2 expression levels were downregulated in the villous of patients with MA. In HTR8/SVneo cells, SKAP2 siRNA silencing regulated the growth and migration, while SKAP2 overexpression promoted growth and migration, and inhibited apoptosis. Additionally, SKAP2 regulated the expression of WAVE2 and ARP2, as well as the colocalization of actin with WAVE2. The SKAP2 W336K mutant could not alter WAVE2 and ARP2 expression, nor HTR8/SVneo cell growth and migration, with or without SKAP2 siRNA transfection. DiscussionSKAP2 could activate the WAVE2-ARP2/3 pathway resulting in an increase of growth and migration in trophoblasts. SKAP2 probably played an important role in MA by affecting the growth and migration of trophoblasts.

Research following genome-wide association study focuses on the multifaceted nature of Src kinase-associated phosphoprotein 2 in type 1 diabetes.

Rutsch et al. identified a patient with type 1 diabetes having a rare Src kinase-associated phosphoprotein 2 variant and investigated the details. As a result, they showed that rare Src kinase-associated phosphoprotein 2 c.475 G>A increases macrophage activity and promotes type 1 diabetes, as well as autoimmune and inflammatory diseases.

SKAP2, a Candidate Gene for Type 1 Diabetes, Regulates β-Cell Apoptosis and Glycemic Control in Newly Diagnosed Patients.

The single nucleotide polymorphism rs7804356 located in the Src kinase-associated phosphoprotein 2 (SKAP2) gene is associated with type 1 diabetes (T1D), suggesting SKAP2 as a causal candidate gene. The objective of the study was to investigate if SKAP2 has a functional role in the β-cells in relation to T1D. In a cohort of children with newly diagnosed T1D, rs7804356 predicted glycemic control and residual β-cell function during the 1st year after diagnosis. In INS-1E cells and rat and human islets, proinflammatory cytokines reduced the content of SKAP2. Functional studies revealed that knockdown of SKAP2 aggravated cytokine-induced apoptosis in INS-1E cells and primary rat β-cells, suggesting an antiapoptotic function of SKAP2. In support of this, overexpression of SKAP2 afforded protection against cytokine-induced apoptosis, which correlated with reduced nuclear content of S536-phosphorylated nuclear factor-κB (NF-κB) subunit p65, lower nitric oxide production, and diminished CHOP expression indicative of decreased endoplasmic reticulum stress. Knockdown of CHOP partially counteracted the increase in cytokine-induced apoptosis caused by SKAP2 knockdown. In conclusion, our results suggest that SKAP2 controls β-cell sensitivity to cytokines possibly by affecting the NF-κB–inducible nitric oxide synthase–endoplasmic reticulum stress pathway.

Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state

There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and –inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen’s d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.

Abstract 001: Skap2 Regulates Atherosclerosis through Macrophage Polarization and Efferocytosis

Background: Atherosclerosis causes more deaths than any other pathophysiologic process. It has a well-established inflammatory, macrophage-mediated component, but potentially protective intracellular macrophage processes in atherosclerosis remain enigmatic. Src Kinase-Associated Phosphoprotein 2 (Skap2) is a macrophage-predominant adaptor protein critical for cytoskeletal reorganization, and thereby, for macrophage migration and chemotaxis. The role of macrophage Skap2 in atherosclerosis is unknown and deserves exploration. Results: Human macrophages express Skap2, and in human arterial gene expression analysis, Skap2 expression is enriched in macrophage-containing areas of human atheroma; the transcript level varies with plaque characteristics, with higher levels in more stable plaques. In ApoE -/- mice on a standard diet, deletion of Skap2 accelerates atherosclerosis by threefold at 18 and 24 weeks. We find that Skap2 expression is switched on only as monocytes differentiate into adherent macrophages, so Skap2 -/- monocytes do not have a defect in infiltrating the atheroma, as reflected by abundant macrophages in the Skap2 -/- plaques. On the other hand, once they fully differentiate, Skap2-deficient macrophages cannot polarize efficiently into alternatively-activated, regulatory macrophages, and instead they preferentially polarize toward the classical pro-inflammatory phenotype both ex vivo and within the developing atheroma. This defect extends to polarized effector functions, as ex vivo analysis of macrophage phagocytosis of dying foam cells indicates that Skap2 is required for the regulatory process of efferocytosis. There are no differences in macrophage proliferation or apoptosis attributable to Skap2. Finally, Skap2 binds to Sirpα, whose interaction with CD47 has been shown to be important for efferocytosis, and we observe colocalization of actin, Sirpα, and CD47 at leading edges of macrophages in a Skap2-dependent manner. Conclusions: Taken together, our findings support a model in which Skap2 drives a regulatory, efferocytic mode of behavior to quell atherosclerosis.

Genome-wide association study and meta-analysis in multiple populations identifies new loci for peanut allergy and establishes C11orf30/EMSY as a genetic risk factor for food allergy

Peanut allergy (PA) is a complex disease with both environmental and genetic risk factors. Previously, PA loci were identified in filaggrin (FLG) and HLA in candidate gene studies, and loci in HLA were identified in a genome-wide association study and meta-analysis. We sought to investigate genetic susceptibility to PA. Eight hundred fifty cases and 926 hyper-control subjects and more than 7.8 million genotyped and imputed single nucleotide polymorphisms (SNPs) were analyzed in a genome-wide association study to identify susceptibility variants for PA in the Canadian population. Ameta-analysis of 2 phenotypes (PA and food allergy) was conducted by using 7 studies from the Canadian, American (n = 2), Australian, German, and Dutch (n = 2) populations. An SNP near integrin α6 (ITGA6) reached genome-wide significance with PA (P = 1.80 × 10-8), whereas SNPs associated with Src kinase-associated phosphoprotein 1 (SKAP1), matrix metallopeptidase 12 (MMP12)/MMP13, catenin α3 (CTNNA3), rho GTPase-activating protein 24 (ARHGAP24), angiopoietin 4 (ANGPT4), chromosome 11 open reading frame (C11orf30/EMSY), and exocyst complex component 4 (EXOC4) reached a threshold suggestive of association (P ≤ 1.49 × 10-6). In the meta-analysis of PA, loci in or near ITGA6, ANGPT4, MMP12/MMP13, C11orf30, and EXOC4 were significant (P ≤ 1.49 × 10-6). When a phenotype of any food allergy was used for meta-analysis, the C11orf30 locus reached genome-wide significance (P = 7.50 × 10-11), whereas SNPs associated with ITGA6, ANGPT4, MMP12/MMP13, and EXOC4 and additional C11orf30 SNPs were suggestive (P ≤ 1.49 × 10-6). Functional annotation indicated that SKAP1 regulates expression of CBX1, which colocalizes with the EMSY protein coded by C11orf30. This study identifies multiple novel loci as risk factors for PA and food allergy and establishes C11orf30 as a risk locus for both PA and food allergy. Multiple genes (C11orf30/EMSY, SKAP1, and CTNNA3) identified by this study are involved in epigenetic regulation of gene expression.

D120 and K152 within the PH Domain of T Cell Adapter SKAP55 Regulate Plasma Membrane Targeting of SKAP55 and LFA-1 Affinity Modulation in Human T Lymphocytes.

The β2-integrin lymphocyte function-associated antigen 1 (LFA-1) is needed for the T cell receptor (TCR)-induced activation of LFA-1 to promote T cell adhesion and interaction with antigen-presenting cells (APCs). LFA-1-mediated cell-cell interactions are critical for proper T cell differentiation and proliferation. The Src kinase-associated phosphoprotein of 55 kDa (SKAP55) is a key regulator of TCR-mediated LFA-1 signaling (inside-out/outside-in signaling). To gain an understanding of how SKAP55 controls TCR-mediated LFA-1 activation, we assessed the functional role of its pleckstrin homology (PH) domain. We identified two critical amino acid residues within the PH domain of SKAP55, aspartic acid 120 (D120) and lysine 152 (K152). D120 facilitates the retention of SKAP55 in the cytoplasm of nonstimulated T cells, while K152 promotes SKAP55 membrane recruitment via actin binding upon TCR triggering. Importantly, the K152-dependent interaction of the PH domain with actin promotes the binding of talin to LFA-1, thus facilitating LFA-1 activation. These data suggest that K152 and D120 within the PH domain of SKAP55 regulate plasma membrane targeting and TCR-mediated activation of LFA-1.

SKAP2 Promotes Podosome Formation to Facilitate Tumor-Associated Macrophage Infiltration and Metastatic Progression.

Tumor-associated macrophages (TAM) play complex and pivotal roles during cancer progression. A subset of metastasis-associated macrophages accumulates within metastatic sites to promote the invasion and growth of tumor cells. Src kinase-associated phosphoprotein 2 (SKAP2), a substrate of Src family kinases, is highly expressed in macrophages from various tumors, but its contribution to the tumor-promoting behavior of TAMs is unknown. Here, we report that SKAP2 regulates podosome formation in macrophages to promote tumor invasion and metastasis. SKAP2 physically interacted with Wiskott-Aldrich syndrome protein (WASP) and localized to podosomes, which were rarely observed in SKAP2-null macrophages. The invasion of peritoneal macrophages derived from SKAP2-null mice was significantly reduced compared with wild-type macrophages, but could be rescued by the restoration of functional SKAP2 containing an intact tyrosine phosphorylation site and the ability to interact with WASP. Furthermore, SKAP2-null mice inoculated with lung cancer cells exhibited markedly decreased lung metastases characterized by reduced macrophage infiltration compared with wild-type mice. Moreover, intravenously injected SKAP2-null macrophages failed to efficiently infiltrate established tumors and promote their growth. Taken together, these findings reveal a novel mechanism by which macrophages assemble the appropriate motile machinery to infiltrate tumors and promote disease progression, and implicate SKAP2 as an attractive candidate for therapeutically targeting TAMs.

The mitosis-regulating and protein-protein interaction activities of astrin are controlled by aurora-A-induced phosphorylation.

Cells display dramatic morphological changes in mitosis, where numerous factors form regulatory networks to orchestrate the complicated process, resulting in extreme fidelity of the segregation of duplicated chromosomes into two daughter cells. Astrin regulates several aspects of mitosis, such as maintaining the cohesion of sister chromatids by inactivating Separase and stabilizing spindle, aligning and segregating chromosomes, and silencing spindle assembly checkpoint by interacting with Src kinase-associated phosphoprotein (SKAP) and cytoplasmic linker-associated protein-1α (CLASP-1α). To understand how Astrin is regulated in mitosis, we report here that Astrin acts as a mitotic phosphoprotein, and Aurora-A phosphorylates Astrin at Ser(115). The phosphorylation-deficient mutant Astrin S115A abnormally activates spindle assembly checkpoint and delays mitosis progression, decreases spindle stability, and induces chromosome misalignment. Mechanistic analyses reveal that Astrin phosphorylation mimicking mutant S115D, instead of S115A, binds and induces ubiquitination and degradation of securin, which sequentially activates Separase, an enzyme required for the separation of sister chromatids. Moreover, S115A fails to bind mitosis regulators, including SKAP and CLASP-1α, which results in the mitotic defects observed in Astrin S115A-transfected cells. In conclusion, Aurora-A phosphorylates Astrin and guides the binding of Astrin to its cellular partners, which ensures proper progression of mitosis.

Spatial and temporal expression of RA70/Scap2 in the developing neural tube

Src kinase-associated phosphoprotein 2 (Ra70/scap2), which was originally isolated as a retinoic acid (RA)-induced gene, associates with molecules that modulate integrin-survival signals. Although RA is essential for vertebrate organogenesis in the posterior region, little is known about the biological role of RA70/Scap2 during development. In the present study, we demonstrate that Ra70/scap2 mRNA is temporally expressed during the RA-induced neuronal differentiation of P19 embryonic carcinoma cells. Homozygous knockout mice in which the Ra70/scap2 gene was replaced with LacZ exhibited embryonic lethality, while heterozygous mice displayed preferential expression of LacZ in posterior neural tissues, including the neural tube and hindbrain during development (E7.5–11.5), but not the forebrain. Ra70/scap2 was expressed in the ependymal layer and ventricular zone in the neural tube, where neuroepithelial cells and neuroblasts with proliferation capacity are localized, respectively. Thus, RA70/Scap2 may be necessary for RA-induced neuronal differentiation from the posterior neuroectoderm.

Regulation of the Src Kinase-associated Phosphoprotein 55 Homologue by the Protein Tyrosine Phosphatase PTP-PEST in the Control of Cell Motility

PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.

The Src Substrate SKAP2 Regulates Actin Assembly by Interacting with WAVE2 and Cortactin Proteins

In our attempt to screen for substrates of Src family kinases in glioblastoma, Src kinase-associated phosphoprotein 2 (SKAP2) was identified. Although SKAP2 has been suggested to be associated with integrin-mediated adhesion of hematopoietic cells, little is known about its molecular function and the effects in other types of cells and tumors. Here, we demonstrate that SKAP2 physically associates with actin assembly factors WAVE2 and cortactin and inhibits their interaction. Cortactin is required for the membrane localization of WAVE2, and SKAP2 suppresses actin polymerization mediated by WAVE2 and cortactin in vitro. Knockdown of SKAP2 in NIH3T3 accelerated cell migration and enhanced translocation of WAVE2 to the cell membrane, and those effects of SKAP2 depend on the binding activity of SKAP2 to WAVE2. Furthermore, reduction of SKAP2 in the glioblastoma promoted tumor invasion both in ex vivo organotypic rat brain slices and immune-deficient mouse brains. These results suggest that SKAP2 negatively regulates cell migration and tumor invasion in fibroblasts and glioblastoma cells by suppressing actin assembly induced by the WAVE2-cortactin complex, indicating that SKAP2 may be a novel candidate for the suppressor of tumor progression.

The Pleckstrin Homology Domain in the SKAP55 Adapter Protein Defines the Ability of the Adapter Protein ADAP To Regulate Integrin Function and NF-κB Activation

Adhesion and degranulation promoting adapter protein (ADAP) is a multifunctional hematopoietic adapter protein that regulates TCR-dependent increases in both integrin function and activation of the NF-κB transcription factor. Activation of integrin function requires both ADAP and the ADAP-associated adapter Src kinase-associated phosphoprotein of 55 kDa (SKAP55). In contrast, ADAP-mediated regulation of NF-κB involves distinct binding sites in ADAP that promote the inducible association of ADAP, but not SKAP55, with the CARMA1 adapter and the TAK1 kinase. This suggests that the presence or absence of associated SKAP55 defines functionally distinct pools of ADAP. To test this hypothesis, we developed a novel SKAP-ADAP chimeric fusion protein and demonstrated that physical association of ADAP with SKAP55 is both sufficient and necessary for the rescue of integrin function in ADAP-deficient T cells. Similar to wild-type ADAP, the SKAP-ADAP chimera associated with the LFA-1 integrin after TCR stimulation. Although the SKAP-ADAP chimera contains the CARMA1 and TAK1 binding sequences from ADAP, expression of the chimera does not restore NF-κB signaling in ADAP(-/-) T cells. A single point mutation in the pleckstrin homology domain of SKAP55 (R131M) blocks the ability of the SKAP-ADAP chimera to restore integrin function and to associate with LFA-1. However, the R131M mutant was now able to restore NF-κB signaling in ADAP-deficient T cells. We conclude that integrin regulation by ADAP involves the recruitment of ADAP to LFA-1 integrin complexes by the pleckstrin homology domain of SKAP55, and this recruitment restricts the ability of ADAP to interact with the NF-κB signalosome and regulate NF-κB activation.

SKAP2, a novel target of HSF4b, associates with NCK2/F-actin at membrane ruffles and regulates actin reorganization in lens cell

In addition to roles in stress response, heat shock factors (HSFs) play crucial roles in differentiation and development. Heat shock transcription factor 4 (HSF4) deficiency leads to defect in lens epithelial cell (LEC) differentiation and cataract formation. However, the mechanism remains obscure. Here, we identified Src kinase-associated phosphoprotein 2 (SKAP2) as a downstream target of HSF4b and it was highly expressed at the anterior tip of lens elongating fibre cells in vivo. The HSF4-deficient lenses showed reduced SKAP2 expression and defects in actin reorganization. The disassembly of stress fibres and formation of cortical actin fibres are critical for the initiation of LEC differentiation. SKAP2 localized at actin-rich ruffles in human LECs (SRA01/04 cells) and knockdown SKAP2 using RNA interference impaired the disassembly of cellular stress fibres in response to fibroblast growth factor (FGF)-b. Overexpression of SKAP2, but not the N-terminal deletion mutant of SKAP2, induced the actin remodelling. We further found that SKAP2 interacted with the SH2 domain of non-catalytic region of tyrosine kinase adaptor protein 2 (NCK2) via its N-terminus. The complex of SKAP2-NCK2-F-actin accumulated at the leading edge of the lamellipodium, where FGF receptors and focal adhesion were also recruited. These results revealed an essential role for HSF4-mediated SKAP2 expression in the regulation of actin reorganization during lens differentiation, likely through a mechanism that SKAP2 anchors the complex of NCK2/focal adhesion to FGF receptors at the lamellipodium in lens epithelial cells.

A novel S3S‐TAP‐tag for the isolation of T‐cell interaction partners of adhesion and degranulation promoting adaptor protein

The identification of modular units of cellular function is a major goal for proteomic research. Protein complexes represent important building blocks defining functionality and deciphering their composition remains a major challenge. Here, we have designed a new tandem affinity purification (TAP) tag (termed S3S-tag) for the isolation of protein complexes. Specifically, the immune cell protein ADAP that regulates integrin adhesion was fused either C- or N-terminally to the S3S-tag. After retroviral transduction of a vector containing S3S-tagged ADAP and internal ribosomal entry site encoded enhanced green fluorescent protein (eGFP), Jurkat T cells were sorted according to eGFP expression and further selected for expression of TAP-tagged protein close to endogenous levels. The combination of a cleavable S-tag and a Strep-tag II allowed for the isolation of ADAP and associated proteins. Subsequently, stable isotope labeling with amino acids in cell culture-based mass spectrometric analysis was performed to identify potentially specific interaction partners. Co-purification of the known interaction partner Src kinase-associated phosphoprotein of 55 kDa indicates the validity of our approach, while the identification of the ENA/VASP family member EVL, the guanine nucleotide exchange factor GEF-H1 and the adaptor protein DOCK2 corroborates a link between ADAP-mediated integrin regulation and the cytoskeleton.

A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation

Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.

Phosphoinositide 3‐kinase‐regulated adapters in lymphocyte activation

Signaling via phosphoinositide 3-kinases (PI3Ks) has emerged as a central component of lymphocyte activation via immunoreceptors, costimulatory receptors, cytokine receptors, and chemokine receptors. The discovery of phosphoinositide-binding pleckstrin homology (PH) domains has substantially increased understanding of how PI3Ks activate cellular responses. Accumulating evidence indicates that PH-domain containing adapter molecules provide important links between PI3K and lymphocyte function. Here, we review data on PI3K-regulated adapter proteins of the Grb-associated binder (GAB), Src kinase-associated phosphoprotein (SKAP), and B-lymphocyte adapter molecule of 32 kDa (Bam32)/ dual-adapter for phosphotyrosine and 3-phosphoinositides (DAPP)/TAPP families, with a focus on the latter group. Current data support the model that recruitment of these adapters to the plasma membrane of activated lymphocytes is driven by the phosphoinositides phosphatidylinositol-3,4,5-tris-phosphate and phosphatidylinositol-3,4-bisphosphate, generated through the action of PI3Ks and under the regulatory control of lipid phosphatases Src homology 2 domain-containing inositol phosphatase (SHIP), phosphatase and tensin homolog, and inositol polyphosphate 4-phosphatase. At the plasma membrane, these adapters serve to assemble distinct protein complexes. Bam32/DAPP1 and SKAPs function to promote activation of monomeric guanosine triphosphatases, including Rac and Rap, and promote integrin activation, lymphocyte adhesion to matrix proteins, and cell:cell interactions between B and T lymphocytes. GABs can provide feedforward amplification or feedback inhibition of PI3K signaling. Current work is further defining the molecular interactions driven by these molecules and identifying the functions of TAPP adapters, which also appear to be involved in lymphocyte adhesion and are specific effectors downstream of the SHIP product phosphatidylinositol-3,4-bisphosphate.