Tyrosine Kinase Adaptor Protein Research Articles

The Oncolytic Avian Reovirus p17 Protein Inhibits Invadopodia Formation in Murine Melanoma Cancer Cells by Suppressing the FAK/Src Pathway and the Formation of theTKs5/NCK1 Complex.

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

NCK1 Modulates Neuronal Actin Dynamics and Promotes Dendritic Spine, Synapse, and Memory Formation.

Memory formation and maintenance is a dynamic process involving the modulation of the actin cytoskeleton at synapses. Understanding the signaling pathways that contribute to actin modulation is important for our understanding of synapse formation and function, as well as learning and memory. Here, we focused on the importance of the actin regulator, noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1), in hippocampal dependent behaviors and development. We report that male mice lacking NCK1 have impairments in both short-term and working memory, as well as spatial learning. Additionally, we report sex differences in memory impairment showing that female mice deficient in NCK1 fail at reversal learning in a spatial learning task. We find that NCK1 is expressed in postmitotic neurons but is dispensable for neuronal proliferation and migration in the developing hippocampus. Morphologically, NCK1 is not necessary for overall neuronal dendrite development. However, neurons lacking NCK1 have lower dendritic spine and synapse densities in vitro and in vivo EM analysis reveal increased postsynaptic density (PSD) thickness in the hippocampal CA1 region of NCK1-deficient mice. Mechanistically, we find the turnover of actin-filaments in dendritic spines is accelerated in neurons that lack NCK1. Together, these findings suggest that NCK1 contributes to hippocampal-dependent memory by stabilizing actin dynamics and dendritic spine formation.SIGNIFICANCE STATEMENT Understanding the molecular signaling pathways that contribute to memory formation, maintenance, and elimination will lead to a better understanding of the genetic influences on cognition and cognitive disorders and will direct future therapeutics. Here, we report that the noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) adaptor protein modulates actin-filament turnover in hippocampal dendritic spines. Mice lacking NCK1 show sex-dependent deficits in hippocampal memory formation tasks, have altered postsynaptic densities, and reduced synaptic density. Together, our work implicates NCK1 in the regulation of actin cytoskeleton dynamics and normal synapse development which is essential for memory formation.

LncRNA NCK1-AS1-mediated regulatory functions in human diseases.

Disease development requires the activation of complex multi-factor processes involving numerous long noncoding RNAs (lncRNAs), which describe non-protein-coding RNAs longer than 200 nucleotides. Emerging evidence indicates that lncRNAs act as essential regulators that perform pivotal roles in the pathogenesis and progression of human diseases. The mechanisms underlying lncRNA involvement in diverse diseases have been extensively explored, and lncRNAs are considered powerful biomarkers for clinical practice. The lncRNA noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) antisense 1 (NCK1-AS1), also known as NCK1 divergent transcript (NCK1-DT), is encoded on human chromosome 3q22.3 and produces a 27,274-base-long transcript. NCK1-AS1 has increasingly been characterized as a causative agent for multiple diseases. The abnormal expression and involvement of NCK1-AS1 in various biological processes have been associated with several diseases. Further exploration of the mechanisms through which NCK1-AS1 contributes to disease development and progression will provide a foundation for potential clinical applications of NCK1-AS1 in the diagnosis and treatment of various diseases. This review summarizes the current understanding of the various functions and mechanisms through which NCK1-AS1 contributes to various diseases and the clinical application prospects for NCK1-AS1.

LncRNA NCK1-AS1 Promotes Malignant Cellular Phenotypes of Laryngeal Squamous Cell Carcinoma via miR-137/NCK1 Axis.

Increasing evidence demonstrates that many long noncoding RNAs (lncRNAs) are implicated with the development of laryngeal squamous cell carcinoma (LSCC). As shown by bioinformatics analysis, lncRNA non-catalytic region of tyrosine kinase adaptor protein 1-antisense 1 (NCK1-AS1) is upregulated in tissues of head and neck squamous cell carcinoma. The study aimed to explore the role and mechanism of NCK1-AS1 in LSCC. NCK1-AS1 expression in LSCC cells was evaluated by reverse transcription qPCR. The viability, proliferation, invasion, migration, and apoptosis of LSCC cells with indicated transfection were evaluated by CCK-8 assays, Ethynyl deoxyuridine incorporation assays, Transwell assays, wound healing assays, and TUNEL assays, respectively. Subcellular fractionation assays were performed to evaluate the cellular distribution of NCK1-AS1 and NCK1. NCK1 protein level in LSCC cells with indicated transfection was quantified by western blotting. The binding relation between miR-137 and NCK1-AS1 (or NCK1) were determined using RNA immunoprecipitation assays and luciferase reporter assays. NCK1-AS1 was highly expressed in LSCC cell lines. NCK1-AS1 depletion suppressed LSCC cell viability, proliferation, invasion, and migration while enhancing cell apoptosis. NCK1, an adjacent gene of NCK1-AS1, is also highly expressed in LSCC cells and was positively regulated by NCK1-AS1. Moreover, NCK1-AS1 interact with miR-137 to upregulate NCK1 expression. NCK1 was the downstream target of miR-137 and was negatively correlated to miR-137. In addition, overexpressed NCK1 reversed the suppressive impact of NCK1-AS1 depletion on malignant behaviors of LSCC cells. NCK1-AS1 contributes to LSCC cellular behaviors by upregulating NCK1 via interaction with miR-137.

Role of the Interaction Between Nck1 and PERK in Pancreatic Beta Cell Function and Survival

Preventing pancreatic β cell failure and death, features of diabetes pathogenesis, could help treat diabetes. PERK, a player in the unfolded protein response (UPR), is essential for β cell function and survival. We have shown that PERK, when residue Y561 is phosphorylated, is bound and regulated by the adaptor protein Nck1 (non-catalytic region of tyrosine kinase adaptor protein 1). Silencing Nck1 in β cells enhances basal PERK activity and prevents its hyperactivation under gluco-lipotoxic conditions, improving proinsulin synthesis and β cell survival. Our preliminary results also showed that islet grafts lacking Nck1 perform better compared to WT grafts to reverse streptozotocin-induced diabetes in mice. Thus, we hypothesize that disruption of the specific Nck1/PERK interaction could improve β cell health and generate a phosphopeptide (TAT-pY561) that sequesters Nck1 and prevents its interaction with PERK. As predicted, this peptide increases insulin synthesis and decreases apoptosis in cultured beta cells under gluco-lipotoxic conditions. In vivo, the peptide administered daily for 3 weeks to mice (prior to and during induction of diabetes using HFD + streptozotocin) increased pancreatic insulin content and glucose-stimulated insulin secretion compared to vehicle treated mice. Moreover, in human diabetic islets, in vitro treatment with the peptide tended to increase insulin content. Our data suggest that inhibition of Nck1/PERK interaction, in mouse and human islets, improves β cell survival and function. Targeting this interaction might prove useful to enhance beta cell health in diabetes and/or when producing sources of beta cells for transplantation.

LncRNA NCK1-AS1 exerts oncogenic property in gastric cancer by targeting the miR-22-3p/BCL9 axis to activate the Wnt/β-catenin signaling.

Long noncoding RNAs (lncRNAs) exert crucial effects on the development of many malignancies, including gastric cancer. Herein, we investigated the role of lncRNA noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) divergent transcript (NCK1-DT, also known as NCK1-AS1) in gastric cancer. Reverse transcription quantitative polymerase chain reaction demonstrated that NCK1-AS1 exhibited high expression in gastric cancer tissues and cells. In vitro assays including MTT, colony formation, Transwell, wound healing and sphere formation assays indicated that NCK1-AS1 depletion inhibited cell proliferation, migration, invasion and stemness maintenance. Luciferase reporter and RIP assays suggested that NCK1-AS1 functioned as a competitive endogenous RNA (ceRNA) for miR-22-3p to positively modulate BCL9 expression. BCL9 was a target gene of miR-22-3p. According to western blot analysis and TOP/FOP flash assay, NCK1-AS1 activated the Wnt/β-catenin signaling via the miR-22-3p/BCL9 axis. Furthermore, rescue experiments verified that NCK1-AS1 affected cellular processes by activating the Wnt/β-catenin signaling pathway via the miR-22-3p/BCL9 axis. Tumor xenograft model validated that NCK1-AS1 promoted tumor growth in vivo via the Wnt/β-catenin signaling by upregulating BCL9 expression. Overall, NCK1-AS1 functions as an oncogene and promotes gastric cancer progression via the miR-22-3p/BCL9-Wnt/β-catenin signaling pathway.

NCK1 Regulates Amygdala Activity to Control Context-dependent Stress Responses and Anxiety in Male Mice

Anxiety disorder (AD) is characterized by the development of maladaptive neuronal circuits and changes to the excitatory/inhibitory (E/I) balance of the central nervous system. Although AD is considered to be heritable, specific genetic markers remain elusive. Recent genome-wide association studies (GWAS) studies have identified non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), a gene that codes for an intracellular adaptor protein involved in actin dynamics, as an important gene in the regulation of mood. Using a murine model in which NCK1 is inactivated, we show that male, but not female, mice display increased levels of context-dependent anxiety-like behaviors along with an increase in circulating serum corticosterone relative to control. Treatment of male NCK1 mutant mice with a positive allosteric modulator of the GABAA receptor rescued the anxiety-like behaviors implicating NCK1 in regulating neuronal excitability. These defects are not attributable to apparent defects in gross brain structure or in axon guidance. However, when challenged in an approach-avoidance conflict paradigm, male NCK1-deficient mice have decreased neuronal activation in the prefrontal cortex (PFC), as well as decreased activation of inhibitory interneurons in the basolateral amygdala (BLA). Finally, NCK1 deficiency results in loss of dendritic spine density in principal neurons of the BLA. Taken together, these data implicate NCK1 in the control of E/I balance in BLA. Our work identifies a novel role for NCK1 in the regulation of sex-specific neuronal circuitry necessary for controlling anxiety-like behaviors. Further, our work points to this animal model as a useful preclinical tool for the study of novel anxiolytics and its significance towards understanding sex differences in anxiolytic function.

Nck1 promotes the progression of ovarian carcinoma by enhancing the PI3K/AKT/p70S6K signaling.

Non-catalytic region of tyrosine kinase adaptor protein 1 (Nck1) is crucial for the progression of cancers. However, little is known on the role of Nck1 in the progression of ovarian carcinoma (OC). Here, we show that Nck1 expression is up-regulated in 176 OC tissues, compared with non-carcinoma ovarian tissues, and the up-regulated Nck1 expression is associated with the aggressiveness of OC and shorter overall and disease-free survival in this population. Higher Nck1 expression was an independent risk factor for poor prognosis of OC. Furthermore, Nck1 silencing by short hairpin RNA (shRNA) technology significantly inhibited the proliferation, migration and invasion of OC cells in vitro and the growth and metastasis of implanted OC tumors in vivo. Human kinase phosphorylation array indicated that Nck1 silencing significantly reduced the relative levels of 11 kinase expression and phosphorylation in OC cells, particularly for decreased levels of p70S6 kinase (p70S6K) and protein kinase B (AKT) expression in SKOV3 cells. Actually, Nck1 silencing significantly decreased PI3K and AKT expression, and reduced AKT and p70S6K phosphorylation while Nck1 over-expression had opposite effects in OC cells. Therefore, our data indicate that Nck1 promotes the progression of OC by enhancing the PI3k/AKT/p70S6K signaling in OC. Our findings suggest that Nck1 expression may be valuable for evaluating the prognosis of OC and as a target for design of new therapies for OC.

FXR Inhibits Endoplasmic Reticulum Stress-Induced NLRP3 Inflammasome in Hepatocytes and Ameliorates Liver Injury

Endoplasmic reticulum (ER) stress is associated withliver injury and fibrosis, and yet the hepatic factors that regulate ER stress-mediated inflammasome activation remain unknown. Here, we report that farnesoid X receptor (FXR) activation inhibits ER stress-induced NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome in hepatocytes. In patients with hepatitis B virus (HBV)-associated hepatic failure or non-alcoholic fatty liver disease, and in mice with liver injury, FXR levels in the liver inversely correlated with the extent of NLRP3 inflammasome activation. Fxr deficiency in mice augmented the ability of ER stress to induceNLRP3 and thioredoxin-interacting protein (TXNIP), whereas FXR ligand activation prevented it, ameliorating liver injury. FXR attenuates CCAAT-enhancer-binding protein homologous protein (CHOP)-dependent NLRP3 overexpression by inhibiting ER stress-mediated protein kinase RNA-like endoplasmic reticulum kinase (PERK) activation. Our findings implicate miR-186 and its target, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), in mediating the inhibition of ER stress by FXR. This study provides the insights on how FXR regulation of ER stress ameliorates hepatocyte death and liver injury and on the molecular basis of NLRP3 inflammasome activation.

A Link Between Alzheimer's and Type II Diabetes Mellitus? Ca+2 -Mediated Signal Control and Protein Localization.

We propose protein localization dependent signal activation (PLDSA) as a model to describe pre-existing protein partitioning between the cytosol, and membrane surface, as a means to modulate signal activation, specificity, and robustness. We apply PLDSA to explain possible molecular links between type II diabetes mellitus (T2DM) and Alzheimer's disease (AD) by describing Ca+2 -mediated interactions between the Src non-receptor tyrosine kinase and p52Shc adaptor protein. We suggest that these interactions may serve as a contributing factor to disease development and progression. In particular, we propose that signaling response is regulated, in part, by Ca+2 -mediated partitioning of lipid-bound and soluble forms of Src and p52shc. Thus, protein-protein interactions that drive signaling in response to extracellular ligand binding are also mediated by partitioning of signaling proteins between membrane-bound and soluble populations. We propose that PLDSA effects may explain, in part, the evolutionary basis of promiscuous protein interaction domains and their importance in cellular function.

Non-catalytic region of tyrosine kinase adaptor protein 2 (NCK2) pathways as factor promoting aggressiveness in ovarian cancer.

In this study we investigated the function of the non-catalytic region of tyrosine kinase adaptor protein 2 (NCK2) and its correlation with ITGB1 and ITGB4 integrins in driving ovarian cancer (OvCa) aggressiveness. We also evaluated whether NCK2 may influence prognosis in OvCa patients. Nanofluidic technology was used to analyze expression of NCK2 in 332 OvCa patients. To evaluate mRNA expression of NCK2, integrins and VEGFA in OvCa cell lines, qRT-PCR was performed. Stable NCK2 overexpression was obtained in OVCAR3. qRT-PCR and Western blot were performed to evaluate expression changes of VEGFA, vimentin, ITGB1, ITGB4, MMP2 and MMP9 under normoxia and hypoxia conditions. Coimmunoprecipitation (Co-IP) was performed in the A2780 cell line to study the interaction between NCK2 and proteins of interest. To investigate whether NCK2 can influence anchorage-independent growth, a soft agar assay was completed. Transwell invasion assay was performed on stable-transfected OVCAR-3 cell lines. Nanofluidic data showed NCK2 can play an important role as a factor promoting tumor aggressiveness and survival in OvCa. This role was also linked to the behaviors of ITGB1 and ITGB4. Moreover, in cells overexpressing NCK2, the expression of vimentin, MMP2, MMP9, VEGFA and ITGB1, but not of ITGB4 was induced by hypoxia. Co-IP showed that NCK2 can directly bind ITGB1, but not VEGFA. NCK2 may be involved in mediating cell-extracellular matrix interactions in OvCa cells by influencing tumor aggressiveness. This study provides evidence of a possible role of NCK2 as biomarker of OvCa progression.

Interactions between hematopoietic progenitor kinase 1 and its adaptor proteins (Review).

Hematopoietic progenitor kinase 1 (HPK1), also known as mitogen‑activated protein kinase kinase kinase kinase 1 is a serine/threonine protein kinase. It is involved in various cellular events, including mitogen‑activated protein kinase signaling, nuclear factor‑κB signaling, cytokine signaling, cellular proliferation and apoptosis, Tcell receptor/B cell receptor signaling and T/B/dendritic cell‑mediated immune responses. Therefore, HPK1 has variety of roles in immunity, and is associated with the pathogenesis of autoimmune diseases, cancer, and the inflammatory response. In these cellular and immune events, HPK1 interacts with several adaptor proteins, including caspase recruitment domain family, member 11, hematopoietic cell‑specific protein1, HPK1‑interacting protein of 55kDa, the growth factor receptor‑bound protein 2 family, linker for activated T‑cells, the SH2 domain‑containing leukocyte protein of 76kDa family, the v‑crk avian sarcoma virus CT10 oncogene homolog family, B‑cell adaptor molecule of 32kDa and non‑catalytic region of tyrosine kinase adaptor protein. These adaptor proteins can couple HPK1 with various effector molecules, leading to the transmission of upstream signals to downstream targets. They are crucial in regulating the relocation, phosphorylation, activation and functions of HPK1. HPK1 can also phosphorylate certain proteins, consequently modulating their functions. This review aims to describe the adaptor proteins, which interact with HPK1, particularly focusing on their modes of interaction with HPK1, and the effects that these interactions cause.

T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells.

BackgroundThe Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated.ResultsTo identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr280 (pTyr280) and pTyr305. These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr280 and pTyr305 on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck.ConclusionsTSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells.

The Tyrosine Kinase Adaptor Protein FRS2 Is Oncogenic and Amplified in High-Grade Serous Ovarian Cancer.

High-grade serous ovarian cancers (HGSOC) are characterized by widespread recurrent regions of copy-number gain and loss. Here, we interrogated 50 genes that are recurrently amplified in HGSOC and essential for cancer proliferation and survival in ovarian cancer cell lines. FRS2 is one of the 50 genes located on chromosomal region 12q15 that is focally amplified in 12.5% of HGSOC. We found that FRS2-amplified cancer cell lines are dependent on FRS2 expression, and that FRS2 overexpression in immortalized human cell lines conferred the ability to grow in an anchorage-independent manner and as tumors in immunodeficient mice. FRS2, an adaptor protein in the FGFR pathway, induces downstream activation of the Ras-MAPK pathway. These observations identify FRS2 as an oncogene in a subset of HGSOC that harbor FRS2 amplifications. These studies identify FRS2 as an amplified oncogene in a subset of HGSOC. FRS2 expression is essential to ovarian cancer cells that harbor 12q15 amplification.

A novel member of B-cell linker protein identified in lamprey, Lampetra japonica.

The B-cell linker protein (BLNK) is an adaptor molecule and plays an important role in signal transduction of B-cell receptor (BCR) and pre-B-cell antigen receptor [1,2]. The BLNK contains a conserved C-terminal Src homology 2 (SH2) domain, a proline-rich region and an N-terminal acidic region [2]. As an adaptor protein, BLNK can bind with some signaling molecules such as Bruton’s tyrosine kinase (BTK), growth factor receptor-bound protein 2 (Grb2), and spleen tyrosine kinases (Syk) through its SH2 domain [3]. When BCR signaling pathway was activated, phosphorylation of BLNK could recruit phospholipase Cg (PLCg), BTK, Grb2, guanine nucleotide exchange factor Vav (Vav) and noncatalytic region of tyrosine kinase adaptor protein (Nck), and regulate downstream signaling pathways [4]. Agnathans, represented by lamprey and hagfish, are the oldest vertebrates currently proved to possess the adaptive immune defenses [5]. Though T-cell receptor and BCR do not exist in jawless vertebrates, recent findings in lamprey have revealed that it possesses an alternative immune system that could specifically recognize and respond to external pathogens [6]. The handling of lamprey (Lampetra japonica) and all experimental procedures were approved by the Animal Welfare and Research Ethics Committee of the Institute of Dalian Medical University (Permit Number: SYXK2004-0029). Adult lampreys were collected from Tongjiang section of the Heilongjiang River (Tongjiang City, China) in December 2012. Adult lampreys (200–220 g in weight) were divided into two groups (20 animals per group): one group animals were immunized with 0.1 mg of LPS (Escherichia coli 0111:B4) (Sigma-Aldrich, St Louis, USA) in 0.1 ml PBS and the control animals were injected with 0.1 ml PBS only. The animals were immunized at 8-day intervals by four intraperitoneal injections. Based on the analysis expressed sequence tags (EST) of the cDNA library constructed with lamprey lymphocyte-like cells by our lab previously, a BLNK ortholog was found using Basic Local Alignment Search Tool (BLAST) in the National Center for Biotechnology Information (http://www. ncbi.nlm.nih.gov/). Total RNA was isolated from lamprey lymphocyte-like cells [7] using RNAiso (TaKaRa, Dalian, China) reagent following the manufacturer instructions, and dissolved in DEPC-treated water and stored at 2808C. First strand 30 and 50 RACE-cDNAs were synthesized from 3 mg of total RNA by Reverse transcriptase M-MLV at 308C for 10 min, 428C for 60 min, 708C for 15 min with the 30-CDS primer and 50-CDS primer and Random 9 mers primer following the manufacturer instructions (TaKaRa). The 30and 50-end sequences of Lj-BLNK were obtained by polymerase chain reaction (PCR) with outer primer, inner primer (TaKaRa) and specific primers (Supplementary Table S1). Taq DNA polymerase (TaKaRa) was used for amplification with the following cycling conditions: 948C for 5 min, followed by 40 amplification cycles at 948C for 30 s, 658C for 30 s, 728C for 2 min and a final extension step at 728C for 10 min. Products were analyzed by electrophoresis in a 2% agarose gel stained with ethidium bromide. The target band of PCR product was isolated and purified, subcloned into a pMD19-T vector using a DNA Ligation kit (TaKaRa) and then subject to DNA sequencing (TaKaRa). Total RNAs were separately extracted from different lamprey tissues including lymphocyte-like cells, gill, heart, liver, intestine, and kidney using RNAiso reagent (TaKaRa), and the total RNAs were treated with DNase I (TaKaRa), and then subject to reverse transcription using PrimeScript RT reagent kit (Perfect Real Time) (TaKaRa). Real-time quantitative PCR experiments were performed with a TaKaRa TP800 Real Time PCR System (TaKaRa) using 2 ml cDNA with 16.8 ml SYBR green PCR mastermix (TaKaRa) and 0.6 ml of each specific primer (Supplementary Table S1). The efficiency of the primers was analyzed in 10-fold serial dilutions of cDNA by calculating the slope of the regression line of the cycle thresholds (Cts) versus the relative concentration of cDNA. The GAPDH of L. japonica was used as an Acta Biochim Biophys Sin 2014, xx: 1–5 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu027.

Src-dependent Tks5 phosphorylation regulates invadopodia-associated invasion in prostate cancer cells.

The Src tyrosine kinase substrate and adaptor protein Tks5 had previously been implicated in the invasive phenotype of normal and transformed cell types via regulation of cytoskeletal structures called podosomes/invadopodia. The role of Src-Tks5 signaling in invasive prostate cancer, however, had not been previously evaluated. We measured the relative expression of Tks5 in normal (n = 20) and cancerous (n = 184, from 92 patients) prostate tissue specimens by immunohistochemistry using a commercially available tumor microarray. We also manipulated the expression and activity of wild-type and mutant Src and Tks5 constructs in the LNCaP and PC-3 prostate cancer cell lines in order to ascertain the role of Src-Tks5 signaling in invadopodia development, matrix-remodeling activity, motility, and invasion. Our studies demonstrated that Src was activated and Tks5 upregulated in high Gleason score prostate tumor specimens and in invasive prostate cancer cell lines. Remarkably, overexpression of Tks5 in LNCaP cells was sufficient to induce invadopodia formation and associated matrix degradation. This Tks5-dependent increase in invasive behavior further depended on Src tyrosine kinase activity and the phosphorylation of Tks5 at tyrosine residues 557 and 619. In PC-3 cells we demonstrated that Tks5 phosphorylation at these sites was necessary and sufficient for invadopodia-associated matrix degradation and invasion. Our results suggest a general role for Src-Tks5 signaling in prostate tumor progression and the utility of Tks5 as a marker protein for the staging of this disease.

Phosphoproteomics of collagen receptor networks reveals SHP-2 phosphorylation downstream of wild-type DDR2 and its lung cancer mutants

Collagen is an important extracellular matrix component that directs many fundamental cellular processes including differentiation, proliferation and motility. The signalling networks driving these processes are propagated by collagen receptors such as the β1 integrins and the DDRs (discoidin domain receptors). To gain an insight into the molecular mechanisms of collagen receptor signalling, we have performed a quantitative analysis of the phosphorylation networks downstream of collagen activation of integrins and DDR2. Temporal analysis over seven time points identified 424 phosphorylated proteins. Distinct DDR2 tyrosine phosphorylation sites displayed unique temporal activation profiles in agreement with in vitro kinase data. Multiple clustering analysis of the phosphoproteomic data revealed several DDR2 candidate downstream signalling nodes, including SHP-2 (Src homology 2 domain-containing protein tyrosine phosphatase 2), NCK1 (non-catalytic region of tyrosine kinase adaptor protein 1), LYN, SHIP-2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2], PIK3C2A (phosphatidylinositol-4-phosphate 3-kinase, catalytic subunit type 2α) and PLCL2 (phospholipase C-like 2). Biochemical validation showed that SHP-2 tyrosine phosphorylation is dependent on DDR2 kinase activity. Targeted proteomic profiling of a panel of lung SCC (squamous cell carcinoma) DDR2 mutants demonstrated that SHP-2 is tyrosine-phosphorylated by the L63V and G505S mutants. In contrast, the I638F kinase domain mutant exhibited diminished DDR2 and SHP-2 tyrosine phosphorylation levels which have an inverse relationship with clonogenic potential. Taken together, the results of the present study indicate that SHP-2 is a key signalling node downstream of the DDR2 receptor which may have therapeutic implications in a subset of DDR2 mutations recently uncovered in genome-wide lung SCC sequencing screens.

FGF-2 prevents cancer cells from ER stress-mediated apoptosis via enhancing proteasome-mediated Nck degradation

Induction of ER (endoplasmic reticulum) stress-mediated apoptosis in cancer cells represents an alternative approach for cancer therapy. Whether FGF-2 (fibroblast growth factor 2)-induced survival signals may interact with ER stress signalling in cancer cells remains elusive. In the present study, we showed that pretreatment with FGF-2 decreased the inhibition of DNA synthesis and induction of apoptosis by two different ER stress inducers, TM (tunicamycin) and TG (thapsigargin), in both human hepatoblastoma HepG2 cells and breast cancer MCF-7 cells. Pretreatment with FGF-2 prevented ER stress-mediated apoptosis by decreasing ER stress-induced CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] expression. We further demonstrated that pretreatment with FGF-2 mediated the decrease in TM-induced CHOP expression and apoptosis through ERK1/2 (extracellular-signal-regulated kinases 1 and 2) pathway. Finally, we demonstrated that FGF-2 promoted proteasome-mediated degradation of Nck (non-catalytic region of tyrosine kinase adaptor protein), an SH (Src homology) 2/SH3-containing adaptor protein. Whereas overexpression of Nck1 decreased FGF-2-induced ERK1/2 phosphorylation to inhibit the effect of FGF-2 on TM-induced CHOP expression and apoptosis, a decrease in Nck expression prevented TM-induced CHOP expression and apoptosis. Taken together, the findings of the present study provide the first evidence that Nck plays a pivotal role in integrating FGF-2 and ER stress signals to counteract the ER stress deleterious effect on cancer cell survival.

SH3-mediated targeting of Wrch1/RhoU by multiple adaptor proteins

Wrch1/RhoU is an atypical member of the Rho family. A major structural difference is the extended N-terminus of Wrch1 (nWrch1) containing three putative SH3 domain-binding motifs whose specificities are unknown. To define the impact of this extended region on coupling Wrch1 to cellular signaling, we analyzed in this study nWrch1 interaction with Src homology 3 (SH3) domains of different adaptor proteins. Using sedimentation and isothermal titration calorimetric (ITC) measurements, we identified isolated SH3 domains of growth factor receptor-bound protein 2 (Grb2), noncatalytic region of tyrosine kinase adaptor protein 1 (Nck1), c-Src, chicken tumor virus no. 10 (CT 10) regulator kinase 1 (Crk1), and p120 as low-affinity Wrch1-binding partners. Interestingly, under cell-based conditions, nWrch1 bound tightly to endogenous Grb2 and Nck, but not to Crk, c-Src, or p120. Consistent with this, a very tight nWrch1 interaction with full-length Grb2 and Nck1 was confirmed in vitro by ITC measurements indicating that high avidity of the adaptor proteins can compensate for the low affinity of their SH3 domains. Peptide analysis revealed that the central PxxP motif of nWrch1, which employs a minimal consensus sequence of eight amino acids with an essential arginine next to the PxxP motif, is responsible for these interactions. Thus, novel functional insights from this study suggest that multiple upstream signals may converge on Wrch1 directly through its SH3 domain-binding properties.

Abstract 503: Investigating the role of Tks5 and invadopodia in prostate tumor progression

Abstract Much of the morbidity and mortality in prostate cancer patients occurs strictly in response to metastatic disease. While ongoing efforts are examining the therapeutic value of Src kinase inhibitors, the mechanism by which Src activates invasive cell behavior within the context of this disease are not well-understood. Invadopodia are cytoskeletal structures formed by cancer cells that enable binding to, proteolytic degradation of, and invasion through the extracellular matrix. We and others have previously demonstrated that the Src tyrosine kinase substrate and adaptor protein Tks5 is important for invadopodia formation and function. Here we demonstrate that Src is activated and Tks5 up-regulated in prostate tumor specimens and invasive cell lines. Remarkably, overexpression of Tks5 alone is sufficient to form invadopodia structures and enhance invadopodia-associated gelatin degradation and invasion in the LNCaP cell line. We further show that this increase in invasive behavior likely depends on Src kinase activity and Tks5 phosphorylation. Our results predict a role for Src-Tks5 signaling in prostate tumor progression. This hypothesis will be tested in future studies using (i) mouse xenografts to monitor tumor development from Tks5-modified prostate cancer cell lines, and (ii) human prostate tumor tissue sections to measure Tks5 staining relative to tumor grade. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 503. doi:1538-7445.AM2012-503