SKAP2 Modular Organization Differently Recognizes SRC Kinases Depending on Their Activation Status and Localization.

Dimerization of SRC kinase adaptor phosphoprotein 2 (SKAP2) induces an increase of binding for most SRC kinases suggesting a fine-tuning with transphosphorylation for kinase activation. This work addresses the molecular basis of SKAP2-mediated SRC kinase regulation through the lens of their interaction capacities. By combining a luciferase complementation assay and extensive site-directed mutagenesis, we demonstrated that SKAP2 interacts with SRC kinases through a modular organization depending both on their phosphorylation-dependent activation and subcellular localization. SKAP2 contains three interacting modules consisting in the dimerization domain, the SRC homology 3 (SH3) domain, and the second interdomain located between the Pleckstrin homology and the SH3 domains. Functionally, the dimerization domain is necessary and sufficient to bind to most activated and myristyl SRC kinases. In contrast, the three modules are necessary to bind SRC kinases at their steady state. The Pleckstrin homology and SH3 domains of SKAP2 as well as tyrosines located in the interdomains modulate these interactions. Analysis of mutants of the SRC kinase family member hematopoietic cell kinase supports this model and shows the role of two residues, Y390 and K7, on its degradation following activation. In this article, we show that a modular architecture of SKAP2 drives its interaction with SRC kinases, with the binding capacity of each module depending on both their localization and phosphorylation state activation. This work opens new perspectives on the molecular mechanisms of SRC kinases activation, which could have significant therapeutic impact.

LYP regulates SLP76 and other adaptor proteins in T cells.

BACKGROUND: The LYP tyrosine phosphatase presents a SNP (1858C > T) that increases the risk of developing autoimmune diseases such as type I diabetes and arthritis. It remains unclear how this SNP affects LYP function and promotes the development of these diseases. The scarce information about LYP substrates is in part responsible for the poor understanding of LYP function. RESULTS: In this study, we identify in T lymphocytes several adaptor proteins as potential substrates targeted by LYP, including FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2. We also show that LYP co-localizes with SLP76 in microclusters, upon TCR engagement. CONCLUSIONS: These data indicate that LYP may modulate T cell activation by dephosphorylating several adaptor proteins, such as FYB, SLP-76, HS-1, Vav, SKAP1 and SKAP2 upon TCR engagement.

Generalized machine learning based on multi-omics data to profile the effect of ferroptosis pathway on prognosis and immunotherapy response in patients with bladder cancer.

INTRODUCTION: Bladder cancer (BLCA) affects millions of people worldwide, with high rates of incidence and mortality. Ferroptosis proves to be a novel form of cell death process that is triggered by oxidative stress. METHODS: We procured a total of 25 single nuclear RNA-seq (snRNA-seq) samples from GSE169379 in GEO database. We obtained different cohorts of BLCA patients from the TCGA and GEO databases for model training and validation. A total of 369 ferroptosis-related genes (FRGs) were selected from the FerrDb database. AUCell analysis was performed to assign ferroptosis scores to all the cell types. Weighted Gene Co-Expression Network Analysis (WGCNA), COX, and LASSO regression analysis were conducted to retain and finalize the genes of prognostic values. Various bioinformatic approaches were utilized to depict immune infiltration profile. We conducted a series of colony formation analysis, flow cytometry and western blot (WB) analysis to determine the role of SKAP1 in BLCA. RESULTS: We divided the cells into high ferroptosis group and low ferroptosis group according to ferroptosis activity score, and then screened 2150 genes most associated with ferroptosis by differential expression analysis, which are related to UV-induced DNA damage, male hormone response, fatty acid metabolism and hypoxia. Subsequently, WGCNA algorithm further screened 741 ferroptosis related genes from the 2150 genes for the construction of prognostic model. Lasso-Cox regression analysis was used to construct the prognostic model, and the prognostic model consisting of 6 genes was obtained, namely JUN, SYT1, MAP3K8, GALNT14, TCIRG1, and SKAP1. Next, we constructed a nomogram model that integrated clinical factors to improving the accuracy. In addition, we performed drug sensitivity analyses in different subgroups and found that Staurosporine, Rapamycin, Gemcitabine, and BI-2536 may be candidates for the drugs treatment in high-risk populations. The ESTIMATE results showed higher stromal scores, immune scores, and ESTIMATE scores in the low-risk group, indicating a higher overall immunity level and immunogenicity of tumor microenvironment (TME) in this group, and tumor immune dysfunction and exclusion (TIDE) analysis confirmed a better response to immunotherapy in the low-risk group. Finally, we selected the oncogene SKAP1 in the prognostic gene for in vitro validation, and found that SKAP1 directly regulated BLCA cell proliferation and apoptosis. CONCLUSION: We identified a set of six genes, JUN, SYT1, MAP3K8, GALNT14, TCIRG1, and SKAP1, that exhibited significant potential in stratification of BLCA patients with varying prognosis. In addition, we uncovered the direct regulatory effect of SKAP1 on BLCA cell proliferation and apoptosis, shedding some light on the role of FRGs in pathogenesis of BLCA.

The association of Plk1 with the astrin-kinastrin complex promotes formation and maintenance of a metaphase plate.

Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The astrin-kinastrin (astrin is also known as SPAG5 and kinastrin as SKAP) complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule-kinetochore attachment. However, the molecular mechanisms by which astrin-kinastrin fulfils these diverse roles are not fully understood. Here, we characterise a direct interaction between astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on astrin as well as four Plk1 phosphorylation sites on astrin. Regulation of astrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression, but promotes stable microtubule-kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule-kinetochore attachment formation, and we suggest that astrin phosphorylation by Plk1 contributes to this process.

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.

SKAP2 as a new regulator of oligodendroglial migration and myelin sheath formation.

Oligodendroglial progenitor cells (OPCs) are highly proliferative and migratory cells, which differentiate into complex myelin forming and axon ensheathing mature oligodendrocytes during myelination. Recent studies indicate that the oligodendroglial cell population is heterogeneous on transcriptional and functional level depending on the location in the central nervous system. Here, we compared intrinsic properties of OPC from spinal cord and brain on functional and transcriptional level. Spinal cord OPC demonstrated increased migration as well as differentiation capacity. Moreover, transcriptome analysis revealed differential expression of several genes between both OPC populations. In spinal cord OPC, we confirmed upregulation of SKAP2, a cytoplasmatic adaptor protein known for its implication in cytoskeletal remodeling and migration in other cell types. Recent findings suggest that actin dynamics determine not only oligodendroglial migration, but also differentiation: Whereas actin polymerization is important for process extension, actin destabilization and depolymerization is required for myelin sheath formation. Downregulation or complete lack of SKAP2 in OPC resulted in reduced migration and impaired morphological maturation in oligodendrocytes. In contrast, overexpression of SKAP2 as well as constitutively active SKAP2 increased OPC migration suggesting that SKAP2 function is dependent on activation by phosphorylation. Furthermore, lack of SKAP2 enhanced the positive effect on OPC migration after integrin activation suggesting that SKAP2 acts as modulator of integrin dependent migration. In summary, we demonstrate the presence of intrinsic differences between spinal cord and brain OPC and identified SKAP2 as a new regulator of oligodendroglial migration and sheath formation.

Dual loss of p110δ PI3-kinase and SKAP (KNSTRN) expression leads to combined immunodeficiency and multisystem syndromic features.

BACKGROUND: We previously reported a novel syndrome characterized by combined immunodeficiency associated with severe developmental defects-subsequently known as Roifman-Chitayat syndrome (RCS; OMIM 613328). Linkage analysis identified 2 disease-associated loci. OBJECTIVES: We sought to identify the genetic defect in these patients and characterize their immunologic cellular abnormalities. METHODS: Genetic, immunologic, protein, and cellular functional analyses were used to identify and characterize patient genetic deficiencies and aberrant patient cell behavior. RESULTS: Deleterious variants were found at both loci identified by linkage analysis: a homozygous stop codon in PI3-kinase p110δ (PIK3CD) and a homozygous frame shift mutation in SKAP (KNSTRN), both ablating protein expression. Patients with RCS display aberrant B-cell development, similar to p110δ-deficient mice, but also aberrant T-cell spreading, cell-cell interaction, and migration. Patients also display significant developmental abnormalities not seen in p110δ knockouts (eg, optic nerve atrophy and skeletal anomalies) that we ascribe to loss of SKAP. Aberrant SKAP expression can prolong anaphase and this may contribute to developmental defects. However, we also identified microtubule-associated protein 4 microtubule-binding protein as a novel SKAP-binding partner and show that it undergoes relocalization in patient T cells, with associated areas of aberrant microtubule hyperstabilization, likely contributing not only to the altered properties of RCS lymphoid cells but also to developmental defects. CONCLUSIONS: The complex RCS presentation, with combined developmental and immunologic defects, is associated with a combined deficiency of 2 genes products, PI3-kinase p110δ and SKAP, both of which appear to play a significant role in the disease.

SKAP1 Expression in Cancer Cells Enhances Colon Tumor Growth and Impairs Cytotoxic Immunity by Promoting Neutrophil Extracellular Trap Formation via the NFATc1/CXCL8 Axis.

The mechanisms underlying the development and progression of colon cancer are not fully understood. Herein, Src kinase associated phosphoprotein 1 (SKAP1), an immune cell adaptor, is identified as a novel colon cancer-related gene. SKAP1 expression is significantly increased in colon cancer cells. High SKAP1 levels are independently predictive of poor survival in patients with colon cancer. Notably, SKAP1 expression in colon cancer cells exerted a significant tumor-promoting effect in vivo rather than in vitro. Screening of tumor-infiltrating immune cells revealed the involvement of neutrophils in SKAP1-induced colon tumor promotion. Enhanced formation of neutrophil extracellular traps (NETs) is found to be a key downstream event that contributed to the pro-tumor role of SKAP1. In colon cancer cells, SKAP1 increased the expression of C-X-C motif chemokine ligand 8 (CXCL8) via nuclear factor of activated T cells c1 (NFATc1). The blockade of CXCL8 or NFATc1 largely attenuated neutrophil infiltration, NET formation, and tumor promotion induced by SKAP1. Furthermore, inhibiting SKAP1-induced NET significantly enhanced the antitumor efficiency of adoptive natural killer cell therapy in colon tumor models. In conclusion, SKAP1 significantly promotes colon cancer growth via the cancer cell/neutrophil NFATc1/CXCL8/NET axis, suggesting that SKAP1 is a potential target for colon cancer therapy.

Drug repositioning for rosacea disease: Biological TARGET identification, molecular docking, pharmacophore mapping, and molecular dynamics analysis.

Rosacea is a chronic dermatological condition that currently lacks a clear treatment approach due to an uncomprehensive knowledge of its pathogenesis. The main obstacle lies in understanding its etiology and the mode of action of the different drugs used. This study aims to clarify these aspects by employing drug repositioning. Using an in silico approach, we performed a transcriptomic analysis comparing samples from individuals with diverse types of rosacea to those from healthy controls to identify genes deregulated in this disease. Subsequently, we realized molecular docking and molecular dynamics studies to assess the binding affinity of drugs currently used to treat rosacea and drugs that target proteins interacting with, and thus affecting, proteins deregulated in rosacea. Our findings revealed that the downregulation of SKAP2 and upregulation of S100A7A in rosacea, could be involved in the pathogenesis of the disease. Furthermore, considering the drugs currently used for rosacea management, we demonstrated stable interactions between isotretinoin and BFH772 with SKAP2, and permethrin and PAC-14028 with S100A7A. Similarly, considering drugs targeting SKAP2 and S100A7A interactome proteins, we found that pitavastatin and dasatinib exert stable interactions with SKAP2, and lovastatin and tirbanibulin with S100A7A. In addition, we determine that the types of bonds involved in the interactions were different in SKAP2 from S100A7A. The drug-SKAP2 interactions are hydrogen bonds, whereas the drug-S100A7A interactions are of the hydrophobic type. In conclusion, our study provides evidence for the possible contribution of SKAP2 and S100A7A to rosacea pathology. Furthermore, it provides significant information on the molecular interactions between drugs and these proteins, highlighting the importance of considering structural features and binding interactions in the design of targeted therapies for skin disorders such as rosacea.

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

Background: The importance of CD11b/CD18 expression in neutrophil effector functions is well known. Beyond KINDLIN3 and TALIN1, which are involved in the induction of the high-affinity binding CD11b/CD18 conformation, the signaling pathways that orchestrate this response remain incompletely understood. Method: 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). Results: 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. Conclusion: 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.

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-A Molecule at the Crossroads for Integrin Signalling and Immune Cell Migration and Function.

Src-kinase associated protein 2 (SKAP2) is an intracellular scaffolding protein that is broadly expressed in immune cells and is involved in various downstream signalling pathways, including, but not limited to, integrin signalling. SKAP2 has a wide range of binding partners and fine-tunes the rearrangement of the cytoskeleton, thereby regulating cell migration and immune cell function. Mutations in SKAP2 have been associated with several inflammatory disorders such as Type 1 Diabetes and Crohn's disease. Rodent studies showed that SKAP2 deficient immune cells have diminished pathogen clearance due to impaired ROS production and/or phagocytosis. However, there is currently no in-depth understanding of the functioning of SKAP2. Nevertheless, this review summarises the existing knowledge with a focus of its role in signalling cascades involved in cell migration, tissue infiltration and immune cell function.

Splicing annotation of endometrial cancer GWAS risk loci reveals potentially causal variants and supports a role for NF1 and SKAP1 as susceptibility genes.

Alternative splicing contributes to cancer development. Indeed, splicing analysis of cancer genome-wide association study (GWAS) risk variants has revealed likely causal variants. To systematically assess GWAS variants for splicing effects, we developed a prioritization workflow using a combination of splicing prediction tools, alternative transcript isoforms, and splicing quantitative trait locus (sQTL) annotations. Application of this workflow to candidate causal variants from 16 endometrial cancer GWAS risk loci highlighted single-nucleotide polymorphisms (SNPs) that were predicted to upregulate alternative transcripts. For two variants, sQTL data supported the predicted impact on splicing. At the 17q11.2 locus, the protective allele for rs7502834 was associated with increased splicing of an exon in a NF1 alternative transcript encoding a truncated protein in adipose tissue and is consistent with an endometrial cancer transcriptome-wide association study (TWAS) finding in adipose tissue. Notably, NF1 haploinsufficiency is protective for obesity, a well-established risk factor for endometrial cancer. At the 17q21.32 locus, the rs2278868 risk allele was predicted to upregulate a SKAP1 transcript that is subject to nonsense-mediated decay, concordant with a corresponding sQTL in lymphocytes. This is consistent with a TWAS finding that indicates decreased SKAP1 expression in blood increases endometrial cancer risk. As SKAP1 is involved in T cell immune responses, decreased SKAP1 expression may impact endometrial tumor immunosurveillance. In summary, our analysis has identified potentially causal endometrial cancer GWAS risk variants with plausible biological mechanisms and provides a splicing annotation workflow to aid interpretation of other GWAS datasets.

SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP-Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore-microtubule attachments to achieve faithful cell division.

Phosphorylation of small kinetochore-associated protein induced by GSK3ß promotes cell migration and invasion in esophageal cancer.

Glycogen synthesis kinase-3ß (GSK-3ß) is a kinase shown to regulate esophageal cancer (EC) progression. However, the significance of GSK-3ß in phosphorylation of small kinetochore-associated protein (SKAP) has not been fully characterized. GSK-3ß/SKAP expression was analyzed in EC tissues by RT-qPCR. The association between GSK-3ß expression and the overall survival was analyzed using the Kaplan-Meier method. Transwell and wound healing assays were performed to assess the effects of GSK-3ß/SKAP knockdown on EC cell migration and invasion. By in vitro kinase assay, the SKAP T294 site was identified as a phosphorylated target of GSK-3ß. Moreover, we established two cell lines expressing either T294D (phosphor-mimic) or T294A (phosphor-deficiency) SKAP to analyze the effect of SKAP phosphorylation on EC cell invasion, migration, and epithelial-mesenchymal transition (EMT) process. GSK-3ß was overexpressed and positively correlated with SKAP levels in EC tissues. Increased GSK-3ß expression was associated with EC poor prognosis. Both of GSK-3ß knockdown and silencing SKAP decreased EC cell migration and invasion. GSK-3ß phosphorylated SKAP protein at Thr294 site. Additionally, a T294D mutant SKAP enhanced cell migration, invasion, and EMT process. Conversely, a T294A mutant SKAP inhibited EC cell malignancy. Meanwhile, cell invasion and migration abilities were inhibited after silencing GSK-3ß in EC109-WT, EC109-T294A and EC109-T294D cells. Phosphorylation of SKAP induced by GSK-3ß promoted EC cell migration and invasion.

The Astrin-SKAP complex reduces friction at the kinetochore-microtubule interface.

The kinetochore links chromosomes to spindle microtubules to drive chromosome segregation at cell division. While we know nearly all mammalian kinetochore proteins, how these give rise to the strong yet dynamic microtubule attachments required for function remains poorly understood. Here, we focus on the Astrin-SKAP complex, which localizes to bioriented kinetochores and is essential for chromosome segregation but whose mechanical role is unclear. Live imaging reveals that SKAP depletion dampens the movement and decreases the coordination of metaphase sister kinetochores and increases the tension between them. Using laser ablation to isolate kinetochores bound to polymerizing versus depolymerizing microtubules, we show that without SKAP, kinetochores move slower on both polymerizing and depolymerizing microtubules and that more force is needed to rescue microtubules to polymerize. Thus, in contrast to the previously described kinetochore proteins that increase the grip on microtubules under force, Astrin-SKAP reduces the grip, increasing attachment dynamics and force responsiveness and reducing friction. Together, our findings suggest a model where the Astrin-SKAP complex effectively "lubricates" correct, bioriented attachments to help preserve them.

THUMPD3-AS1 facilitates cell growth and aggressiveness by the miR-218-5p/SKAP1 axis in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a malignant cancer with a high mortality. Accumulating studies have revealed that mRNAs involved in ceRNA (competing endogenous RNA) network are implicated in the tumorigenesis and development of CRC. Here, we aimed to elucidate the ceRNA network involving Src kinase associated phosphoprotein 1 (SKAP1) in the biological characteristics of CRC. METHODS: Expression levels of genes in colon adenocarcinoma (COAD) samples and prognosis of COAD patients were predicted using publicly available online tool. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clony formation and Transwell assays were conducted to test the biological functions of SKAP1 and THUMPD3 antisense RNA 1 (THUMPD3-AS1) in CRC cells. Western blot was used to measure the protein levels of SKAP1. Gene expression in CRC cells was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR). The interaction between miR-218-5p and THUMPD3-AS1 (or SKAP1) was verified by RNA pulldown and luciferase reporter assays. RESULTS: SKAP1 was upregulated in COAD tissues and CRC cells and it reflected a poor prognosis in patients with COAD. SKAP1 knockdown inhibited CRC (HT-29 and HCT-116) cell proliferation, migration and invasion. Mechanistically, THUMPD3-AS1 acted as a ceRNA to sponge miR-218-5p and subsequently upregulated SKAP1 expression in CRC cells. SKAP1 overexpression reversed the suppressive effect of THUMPD3-AS1 knockdown on proliferation, migration and invision of CRC cells. CONCLUSIONS: THUMPD3-AS1 promotes CRC cell growth and aggressiveness by regulating the miR-218-5p/SKAP1 axis.

The Multiple Roles of the Cytosolic Adapter Proteins ADAP, SKAP1 and SKAP2 for TCR/CD3 -Mediated Signaling Events.

T cells are the key players of the adaptive immune response. They coordinate the activation of other immune cells and kill malignant and virus-infected cells. For full activation T cells require at least two signals. Signal 1 is induced after recognition of MHC/peptide complexes presented on antigen presenting cells (APCs) by the clonotypic TCR (T-cell receptor)/CD3 complex whereas Signal 2 is mediated via the co-stimulatory receptor CD28, which binds to CD80/CD86 molecules that are present on APCs. These signaling events control the activation, proliferation and differentiation of T cells. In addition, triggering of the TCR/CD3 complex induces the activation of the integrin LFA-1 (leukocyte function associated antigen 1) leading to increased ligand binding (affinity regulation) and LFA-1 clustering (avidity regulation). This process is termed "inside-out signaling". Subsequently, ligand bound LFA-1 transmits a signal into the T cells ("outside-in signaling") which enhances T-cell interaction with APCs (adhesion), T-cell activation and T-cell proliferation. After triggering of signal transducing receptors, adapter proteins organize the proper processing of membrane proximal and intracellular signals as well as the activation of downstream effector molecules. Adapter proteins are molecules that lack enzymatic or transcriptional activity and are composed of protein-protein and protein-lipid interacting domains/motifs. They organize and assemble macromolecular complexes (signalosomes) in space and time. Here, we review recent findings regarding three cytosolic adapter proteins, ADAP (Adhesion and Degranulation-promoting Adapter Protein), SKAP1 and SKAP2 (Src Kinase Associated Protein 1 and 2) with respect to their role in TCR/CD3-mediated activation, proliferation and integrin regulation.

SKAP2 is required for defense against K. pneumoniae infection and neutrophil respiratory burst.

Klebsiella pneumoniae is a respiratory, blood, liver, and bladder pathogen of significant clinical concern. We show that the adaptor protein, SKAP2, is required for protection against K. pneumoniae (ATCC 43816) pulmonary infections. Skap2-/- mice had 100-fold higher bacterial burden when compared to wild-type and burden was controlled by SKAP2 expression in innate immune cells. Skap2-/- neutrophils and monocytes were present in infected lungs, and the neutrophils degranulated normally in response to K. pneumoniae infection in mice; however, K. pneumoniae-stimulated reactive oxygen species (ROS) production in vitro was abolished. K. pneumoniae-induced neutrophil ROS response required the activity of SFKs, Syk, Btk, PLCγ2, and PKC. The loss of SKAP2 significantly hindered the K. pneumoniae-induced phosphorylation of SFKs, Syk, and Pyk2 implicating SKAP2 as proximal to their activation in pathogen-signaling pathways. In conclusion, SKAP2-dependent signaling in neutrophils is essential for K. pneumoniae-activated ROS production and for promoting bacterial clearance during infection.

Klebsiella pneumoniae is a type of bacteria that can cause life-threatening infections ­ including pneumonia, blood stream infections, and urinary tract infections ­ in hospitalized patients. These infections can be difficult to treat because some K. pneumoniae are resistant to antibiotics. The bacteria are normally found in the human intestine, and they do not usually cause infections in healthy people. This implies that healthy people's immune systems are better able to fend off K. pneumoniae infections; learning how could help scientists develop new ways to treat or prevent infections in hospitalized patients. In healthy people, a type of immune cell called neutrophils are the first line of defense against bacterial infections. Several different proteins are needed to activate neutrophils, including a protein called SKAP2. But the role of this protein in fighting K. pneumoniae infections is not clear. To find out what role SKAP2 plays in the defense against pneumonia caused by K. pneumoniae, Nguyen et al. compared infections in mice with and without the protein. Mice lacking SKAP2 in their white blood cells had more bacteria in their lungs than normal mice. The experiments showed that neutrophils from mice with SKAP2 produce a burst of chemicals called "reactive oxygen species", which can kill bacteria. But neutrophils without the protein do not. Without SKAP2, several proteins that help produce reactive oxygen species do not work. Understanding the role of SKAP2 in fighting infections may help scientists better understand the immune system. This could help clinicians to treat conditions that cause it to be hyperactive or ineffective. More studies are needed to determine if SKAP2 works the same way in human neutrophils and if it works against all types of K. pneumoniae. If it does, then scientists might be able use this information to develop therapies that help the immune system fight infections.

Unraveling neutrophil- Yersinia interactions during tissue infection.

The human and animal pathogens Yersinia pestis, which causes bubonic and pneumonic plague, and Yersinia pseudotuberculosis and Yersinia enterocolitica, which cause gastroenteritis, share a type 3 secretion system which injects effector proteins, Yops, into host cells. This system is critical for virulence of all three pathogens in tissue infection. Neutrophils are rapidly recruited to infected sites and all three pathogens frequently interact with and inject Yops into these cells during tissue infection. Host receptors, serum factors, and bacterial adhesins appear to collaborate to promote neutrophil- Yersinia interactions in tissues. The ability of neutrophils to control infection is mixed depending on the stage of infection and points to the efficiency of Yops and other bacterial factors to mitigate bactericidal effects of neutrophils. Yersinia in close proximity to neutrophils has higher levels of expression from yop promoters, and neutrophils in close proximity to Yersinia express higher levels of pro-survival genes than migrating neutrophils. In infected tissues, YopM increases neutrophil survival and YopH targets a SKAP2/SLP-76 signal transduction pathway. Yet the full impact of these and other Yops and other Yersinia factors on neutrophils in infected tissues has yet to be understood.