Seeking a better understanding of the non-receptor tyrosine kinase, SRMS.

SRMS (Src-Related kinase lacking C-terminal regulatory tyrosine and N-terminal Myristoylation Sites) is a non-receptor tyrosine kinase first reported in a 1994 screen for genes regulating murine neural precursor cells. SRMS, pronounced "Shrims", lacks the C-terminal regulatory tyrosine critical for the regulation of the enzymatic activity of Src-family kinases (SFKs). Another remarkable characteristic of SRMS is its localization into distinct SRMS cytoplasmic punctae (SCPs) or GREL (Goel Raghuveera-Erique Lukong) bodies, a pattern not observed in the SFKs. This unique subcellular localization of SRMS could dictate its cellular targets, proteome, and potentially, substrates. However, the function of SRMS is still relatively unknown. Further, how is its activity regulated and by what cellular targets? Studies have emerged highlighting the potential role of SRMS in autophagy and in regulating the activation of BRK/PTK6. Potential novel cellular substrates have also been identified, including DOK1, vimentin, Sam68, FBKP51, and OTUB1. Recent studies have also demonstrated the potential role of the kinase in various cancers, including gastric and colorectal cancers and platinum resistance in ovarian cancer. This review discusses the advancements made in SRMS-related biology to date and the path to understanding the cellular and physiological significance of the kinase.

An Integrated Proteomic Strategy to Identify SHP2 Substrates.

Protein phosphatases play an essential role in normal cell physiology and the development of diseases such as cancer. The innate challenges associated with studying protein phosphatases have limited our understanding of their substrates, molecular mechanisms, and unique functions within highly coordinated networks. Here, we introduce a novel strategy using substrate-trapping mutants coupled with quantitative proteomics methods to identify physiological substrates of Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) in a high-throughput manner. The technique integrates three parallel mass spectrometry-based proteomics experiments, including affinity isolation of substrate-trapping mutant complex using wild-type and SHP2 KO cells, in vivo global quantitative phosphoproteomics, and in vitro phosphatase reaction. We confidently identified 18 direct substrates of SHP2 in the epidermal growth factor receptor signaling pathways, including both known and novel SHP2 substrates. Docking protein 1 was further validated using biochemical assays as a novel SHP2 substrate, providing a mechanism for SHP2-mediated Ras activation. This advanced workflow improves the systemic identification of direct substrates of protein phosphatases, facilitating our understanding of the equally important roles of protein phosphatases in cellular signaling.

Analysis of the DOK1 gene in breast cancer.

Breast cancer, which is the most common type of cancer among women, is a heterogenous disease. It results from progressive accumulation of genetic and epigenetic alterations in different genes. The Dok1 protein has been identified as the major substrate of protein tyrosine kinases in hematopoietic cells. It is considered as a tumor suppressor due to the reports which describe its inhibitory effect on major oncogenic signaling pathways such as Mek/Erk/PI3k/Akt and Wnt/ß-catenin. In this study, we investigated the mutation frequency of the DOK1 gene in 118 breast tumors using Sanger sequencing and DOK1 mRNA expression level in 63 breast cancer samples using qRT-PCR methods. Although the mutation frequency was low DOK1 mRNA expression levels were significantly reduced (63.5%) in the tumors compared to adjacent non-cancerous tissue. We also correlated expression changes with clinicopathological characteristics. Low mRNA levels correlated with age (p = 0.01) and c-erbB-2 (p = 0.05). In most of the previous reports, down-regulation of DOK1 mRNA expression has been associated with promoter methylation. We identified four different coding sequence alterations in 5.1% (6/118) of the tumor samples. However, all of these alterations were located in the functional domains of the protein. Therefore, these mutations may affect the function and/or cellular localization of the protein and contribute to cancer progression by this way. In conclusion our data indicate that DOK1 acts as a tumor suppressor in breast cancer and association of Dok1 with the c-erbB-2 mediated mechanism of action in breast cancer needs to be investigated.

Docking protein-1 promotes inflammatory macrophage signaling in gastric cancer.

Docking protein-1 (DOK1) is a tumor suppressor frequently lost in malignant cells, however, it retains the ability to control activities of immune receptors in adjacent stroma cells of the tumor microenvironment. We therefore hypothesized that addressing DOK1 may be useful for cancer immunotherapy. DOK1 mRNA and DOK1 protein expression were downregulated in tumor cells of gastric cancer patients (n = 249). Conversely, its expression was up-regulated in cases positive for Epstein Barr Virus (EBV+) together with genes related to macrophage biology and targets of clinical immunotherapy such as programmed-cell-death-ligand-1 (PD-L1). Notably, high DOK1 positivity in stroma cells conferred poor prognosis in patients and correlated with high levels of inducible nitric oxide synthase in CD68+ tumor-associated macrophages. In macrophages derived from human monocytic leukemia cell lines, DOK1 (i) was inducible by agonists of the anti-diabetic transcription factor peroxisome proliferator-activated receptor-gamma (PPARγ), (ii) increased polarization towards an inflammatory phenotype, (iii) augmented nuclear factor-κB-dependent transcription of pro-inflammatory cytokines and (iv) reduced PD-L1 expression. These properties empowered DOK1+ macrophages to decrease the viability of human gastric cancer cells in contact-dependent co-cultures. DOK1 also reduced PD-L1 expression in human primary blood monocytes. Our data propose that the drugability of DOK1 may be exploited to reprogram myeloid cells and enforce the innate immune response against EBV+ human gastric cancer.

Nck adapter proteins promote podosome biogenesis facilitating extracellular matrix degradation and cancer invasion.

BACKGROUND: Podosomes are membrane-bound adhesive structures formed by actin remodeling. They are capable of extracellular matrix (ECM) degradation, which is a prerequisite for cancer cell invasion and metastasis. The signaling mechanism of podosome formation is still unknown in cancer. We previously reported that Nck adaptors regulate directional cell migration and endothelial lumen formation by actin remodeling, while deficiency of Nck reduces cancer metastasis. This study evaluated the role of Nck adaptors in podosome biogenesis and cancer invasion. METHODS: This study was conducted in vitro using both healthy cells (Human Umbilical Vein Endothelial Cell, 3T3 fibroblasts) and cancer cells (prostate cancer cell line; PC3, breast cancer cell line; MDA-MB-231). Confocal and TIRF imaging of cells expressing Green Fluorescence Protein (GFP)  mutant under altered levels of Nck or downstream of kinase 1 (Dok1) was used to evaluate the podosome formation and fluorescent gelatin matrix degradation. Levels of Nck in human breast carcinoma tissue sections were detected by immune histochemistry using Nck polyclonal antibody. Biochemical interaction of Nck/Dok1 was detected in podosome forming cells using immune precipitation and far-western blotting. RESULTS: This study demonstrates that ectopic expression of Nck1 and Nck2 can induce the endothelial podosome formation in vitro. Nck silencing by short-hairpin RNA blocked podosome biogenesis and ECM degradation in cSrc-Y530F transformed endothelial cells in this study. Immunohistochemical analysis revealed the Nck overexpression in human breast carcinoma tissue sections. Immunoprecipitation and far-western blotting revealed the biochemical interaction of Nck/p62Dok in podosome forming cells. CONCLUSIONS: Nck adaptors in interaction with Dok1 induce podosome biogenesis and ECM degradation facilitating cancer cell invasion, and therefore a bona fide target of cancer therapy.

Talin promotes integrin activation accompanied by generation of tension in talin and an increase in osmotic pressure in neurite outgrowth.

Neuronal polarization depends on the interaction of intracellular chemical and mechanical activities in which the cytoplasmic protein, talin, plays a pivotal role during neurite growth. To better understand the mechanism underlying talin function in neuronal polarization, we overexpressed several truncated forms of talin and found that the presence of the rod domain within the overexpressed talin is required for its positive effect on neurite elongation because the neurite number only increased when the talin head region was overexpressed. The tension in the talin rod was recognized using a Förster resonance energy transfer-based tension probe. Nerve growth factor treatment resulted in inward tension of talin elicited by microfilament force and outward osmotic pressure. By contrast, the glial scar-inhibitor aggrecan weakened these forces, suggesting that interactions between inward pull forces in the talin rod and outward osmotic pressure participate in neuronal polarization. Integrin activation is also involved in up-regulation of talin tension and osmotic pressure. Aggrecan stimuli resulted in up-regulation of docking protein 1 (DOK1), leading to the down-regulation of integrin activity and attenuation of the intracellular mechanical force. Our study suggests interactions between the intracellular inward tension in talin and the outward osmotic pressure as the effective channel for promoting neurite outgrowth, which can be up-regulated by integrin activation and down-regulated by DOK1.-Wang, Y., Zhang, X., Tian, J., Shan, J., Hu, Y., Zhai, Y., Guo, J. Talin promotes integrin activation accompanied by generation of tension in talin and an increase in osmotic pressure in neurite outgrowth.

Interaction Analyses of 14-3-3ζ, Dok1, and Phosphorylated Integrin ß Cytoplasmic Tails Reveal a Bi-molecular Switch in Integrin Regulation.

Integrins are hetero-dimeric (α and ß subunits) type I transmembrane proteins that facilitate cell adhesion and migration. The cytoplasmic tails (CTs) of integrins interact with a plethora of intra-cellular proteins that are required for integrin bidirectional signaling. In particular, the ß CTs of integrins are known to recruit a variety of cytosolic proteins that often have overlapping recognition sites. However, the chronological sequence of ß CTs/cytosolic proteins interactions remains to be fully characterized. Previous studies have shown that the scaffold protein 14-3-3ζ binds to phosphorylated ß CTs in activated integrins, whereas interactions of Dok-1 with phosphorylated ß CTs maintained integrins in the resting state. In this study, we examined the binding interactions between 14-3-3ζ, Dok1, and phosphorylated integrin ß2 and ß3 CTs. We show that the scaffold protein 14-3-3ζ interacts with the phosphotyrosine binding (PTB) domain of Dok1 even in the absence of the phosphorylated integrin ß CTs. The interactions were mapped onto the ß-sheet region of the PTB domain of Dok1. Furthermore, we provide evidence that the 14-3-3ζ/Dok1 binary complex is able to bind to their cognate phosphorylated sequence motifs in the integrin ß CTs. We demonstrate that Thr phosphorylated pTTT ß2 CT or pTST ß3 CT can bind to 14-3-3ζ that is in complex with the Dok1 PTB domain, whereas Ser phosphorylated ß2 CT or Tyr phosphorylated ß3 CT interacted with Dok1 in 14-3-3ζ/Dok1 complex. Based on these data, we propose that 14-3-3ζ/Dok1 complex could serve as a molecular switch providing novel molecular insights into the regulating integrin activation.

Key Players of Cisplatin Resistance: Towards a Systems Pharmacology Approach.

The major obstacle in the clinical use of the antitumor drug cisplatin is inherent and acquired resistance. Typically, cisplatin resistance is not restricted to a single mechanism demanding for a systems pharmacology approach to understand a whole cell's reaction to the drug. In this study, the cellular transcriptome of untreated and cisplatin-treated A549 non-small cell lung cancer cells and their cisplatin-resistant sub-line A549rCDDP2000 was screened with a whole genome array for relevant gene candidates. By combining statistical methods with available gene annotations and without a previously defined hypothesis HRas, MAPK14 (p38), CCL2, DOK1 and PTK2B were identified as genes possibly relevant for cisplatin resistance. These and related genes were further validated on transcriptome (qRT-PCR) and proteome (Western blot) level to select candidates contributing to resistance. HRas, p38, CCL2, DOK1, PTK2B and JNK3 were integrated into a model of resistance-associated signalling alterations describing differential gene and protein expression between cisplatin-sensitive and -resistant cells in reaction to cisplatin exposure.

Notch ligand Delta-like1 enhances degranulation and cytokine production through a novel Notch/Dok-1/MAPKs pathway in vitro.

Food allergy includes sensitization phase and effect phase, and effect cells degranulate and secrete cytokines in the effect phase, causing allergic clinical symptoms. We have demonstrated that Notch signaling plays an important role in the sensitization phase, but its role in effect phases still remains unclear. In this study, we investigated the role of Notch signaling in degranulation and cytokine production of the effect phase response. A RBL-2H3 cell model was used and Notch signaling was induced by priming with Notch ligands. Our results showed after priming with Notch ligand, Delta-like1(Dll1)-Fc, ß-hexosaminidase release, and cytokines production, including TGF-ß, IL-1ß, IL-4, IL-6, and IL-13, were increased significantly, and the enhancement was abolished after DAPT treatment, a γ-secretase inhibitor, indicating that Dll1 Notch signaling enhanced RBL-2H3 cell degranulation and cytokine production. Western blot analysis showed that Dll1 Notch signaling augmented high-affinity IgE receptors-mediated phosphorylation of MAPKs through suppressing the expression of downstream tyrosine kinases 1 (Dok-1). Besides, a passive systemic anaphylaxis mouse model was used to confirm the role of Notch signaling. And our data showed that allergic clinical features of mice were alleviated, and the level of degranulation was decreased significantly after inhibiting Notch signaling in vivo. Therefore, we demonstrated Notch ligand Dll1 enhanced RBL-2H3 cell degranulation and cytokine production through a novel Notch/Dok-1/MAPKs pathway, suggesting Notch signaling played a key role in the effect phase of food allergy.

Methylation-associated DOK1 and DOK2 down-regulation: Potential biomarkers for predicting adverse prognosis in acute myeloid leukemia.

DOK-1 and DOK-2 (DOK1/2) are closely related members of downstream of tyrosine kinase (DOK) family genes, which are found to be frequently rearranged in several hematopoietic cancers. However, the clinical implications of DOK1/2 in acute myeloid leukemia (AML) remain largely unknown. To investigate the clinical significance, real-time quantitative PCR (RQ-PCR) was carried out to detect DOK1/2 expressions in 125 de novo AML patients and 28 healthy controls. Real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP) were applied to detect DOK1/2 methylation level and density. DOK1/2 expressions were significantly down-regulated in AML patients. The promoters of DOK1/2 were highly hypermethylated and negatively correlated with DOK1/2 expressions in AML patients. In addition, we also confirmed that DOK1/2 expressions could be restored by DOK1/2 demethylation using 5-aza-2'-deoxycytidine in leukemia cell line THP-1. Survival analyses showed that low-expressed DOK1/2 were associated with markedly shorter overall survival and leukemia free survival in both whole-cohort AML and non-M3 AML patients. Multivariate analyses further revealed that DOK1/2 were act as independent prognostic factors in AML patients. These findings indicate that decreased DOK1/2 expressions associated with their promoter hypermethylations predict adverse prognosis in AML.

Expression of DOK1, 2, and 3 genes in HTLV-1-infected T cells.

Human T-cell leukemia virus type 1 (HTLV-1) can cause an aggressive malignancy known as adult T-cell leukemia/lymphoma (ATLL). The Tax protein encoded by the pX region of the HTLV-1 genome appears to be a key element in the early stage of ATLL development. In this study, we examined the expression of the downstream of tyrosine kinase (DOK) family members DOK1, DOK2 and DOK3, recently reported to be tumor suppressors, in HTLV-1-transformed T cells (MT-2 and HUT-102) and TL-Om1 cells derived from ATLL leukemic cells. DOK2 and DOK3 expression was significantly reduced in MT-2, HUT-102, and TL-Om1 cells compared with their expression in uninfected T cells, and the expression of DOK3 was reduced by the induction of Tax expression in T cells.

Photoactivation of Dok1/ERK/PPARγ signaling axis inhibits excessive lipolysis in insulin-resistant adipocytes.

Insulin resistance is a hallmark of the metabolic syndrome and type 2 diabetes. Increased plasma FFA level is an important cause of obesity-associated insulin resistance. Over-activated ERK is closely related with FFA release from adipose tissues in patients with type 2 diabetes. Nevertheless, there are no effective strategies to lower plasma FFA level. Low-power laser irradiation (LPLI) has been reported to regulate multiple biological processes. However, whether LPLI could ameliorate metabolic disorders and the molecular mechanisms involved remain unknown. In this study, we first demonstrated that LPLI suppresses excessive lipolysis of insulin-resistant adipocytes by activating tyrosine kinases-1(Dok1)/ERK/PPARγ pathway. Our data showed that LPLI inhibits ERK phosphorylation through the activation of Dok1, resulting in decreased phospho-PPARγ level. Non-phosphorylated PPARγ maintains in nucleus to promote the expression of adipogenic genes, reversing excessive lipolysis in insulin-resistant adipocytes. In summary, the present research highlights the important roles of Dok1/ERK/PPARγ pathway in lowering FFA release from adipocytes, and our research extends the knowledge of the biological effects induced by LPLI.

A crucial role for DOK1 in PDGF-BB-stimulated glioma cell invasion through p130Cas and Rap1 signalling.

DOK1 regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility. Mechanisms regulating tumour cell motility are essential for invasion and metastasis. We report here that PDGF-BB-mediated glioma cell invasion and migration are dependent on the adaptor protein downstream of kinase 1 (DOK1). DOK1 is expressed in several glioma cell lines and in tumour biopsies from high-grade gliomas. DOK1 becomes tyrosine phosphorylated upon PDGF-BB stimulation of human glioma cells. Knockdown of DOK1 or expression of a DOK1 mutant (DOK1FF) containing Phe in place of Tyr at residues 362 and 398, resulted in inhibition of both the PDGF-BB-induced tyrosine phosphorylation of p130Cas (also known as BCAR1) and the activation of Rap1. DOK1 colocalises with tyrosine phosphorylated p130Cas at the cell membrane of PDGF-BB-treated cells. Expression of a non-tyrosine-phosphorylatable substrate domain mutant of p130Cas (p130Cas15F) inhibited PDGF-BB-mediated Rap1 activation. Knockdown of DOK1 and Rap1 inhibited PDGF-BB-induced chemotactic cell migration, and knockdown of DOK1 and Rap1 and expression of DOK1FF inhibited PDGF-mediated three-dimensional (3D) spheroid invasion. These data show a crucial role for DOK1 in the regulation of PDGF-BB-mediated tumour cell motility through a p130Cas-Rap1 signalling pathway. [Corrected]

The unique N-terminal region of SRMS regulates enzymatic activity and phosphorylation of its novel substrate docking protein 1.

SRMS (Src-related tyrosine kinase lacking C-terminal regulatory tyrosine and N-terminal myristoylation sites) belongs to a family of nonreceptor tyrosine kinases, which also includes breast tumour kinase and Fyn-related kinase. SRMS, similar to breast tumour kinase and Fyn-related kinase, harbours a Src homology 3 and Src homology 2, as well as a protein kinase domain. However, unlike breast tumour kinase and Fyn-related kinase, SRMS lacks a C-terminal regulatory tail but distinctively possesses an extended N-terminal region. Both breast tumour kinase and Fyn-related kinase play opposing roles in cell proliferation and signalling. SRMS, however, is an understudied member of this family. Although cloned in 1994, information on the biochemical, cellular and physiological roles of SRMS remains unreported. The present study is the first to explore the expression pattern of SRMS in breast cancers, its enzymatic activity and autoregulatory elements, and the characterization of docking protein 1 as its first bonafide substrate. We found that, similar to breast tumour kinase, SRMS is highly expressed in most breast cancers compared to normal mammary cell lines and tissues. We generated a series of SRMS point and deletion mutants and assessed enzymatic activity, subcellular localization and substrate recognition. We report for the first time that ectopically-expressed SRMS is constitutively active and that its N-terminal region regulates the enzymatic activity of the protein. Finally, we present evidence indicating that docking protein 1 is a direct substrate of SRMS. Our data demonstrate that, unlike members of the Src family, the enzymatic activity of SRMS is regulated by the intramolecular interactions involving the N-terminus of the enzyme and that docking protein 1 is a bona fide substrate of SRMS.

Molecular mechanisms of glucocorticoid action in mast cells.

Glucocorticoids are compounds that have successfully been used over the years in the treatment of inflammatory disorders. They are known to exhibit their effects through the glucocorticoid receptor (GR) that acts to downregulate the action of proinflammatory transcription factors such as AP-1 and NF-κB. The GR also exerts anti-inflammatory effects through activation of distinct genes. In addition to their anti-inflammatory actions, glucocorticoids are also potent antiallergic compounds that are widely used in conditions such as asthma and anaphylaxis. Nevertheless the mechanism of action of this hormone in these disorders is not known. In this article, we have reviewed reports on the effects of glucocorticoids in mast cells, one of the important immune cells in allergy. Building on the knowledge of the molecular action of glucocorticoids and the GR in the treatment of inflammation in other cell types, we have made suggestions as to the likely mechanisms of action of glucocorticoids in mast cells. We have further identified some important questions and research directions that need to be addressed in future studies to improve the treatment of allergic disorders.