Kisspeptin-10 binding to Gpr54 in osteoclasts prevents bone loss by activating Dusp18-mediated dephosphorylation of Src.

Osteoclasts are over-activated as we age, which results in bone loss. Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCRs) are the targets for ∼35% of approved drugs but it is still unclear how GPCRs regulate Src kinase activity. Here, we reveal that GPR54 activation by its natural ligand Kisspeptin-10 (Kp-10) causes Dusp18 to dephosphorylate Src at Tyr 416. Mechanistically, Gpr54 recruits both active Src and the Dusp18 phosphatase at its proline/arginine-rich motif in its C terminus. We show that Kp-10 binding to Gpr54 leads to the up-regulation of Dusp18. Kiss1, Gpr54 and Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss, and Kp-10 abrogated bone loss by suppressing osteoclast activity in vivo. Therefore, Kp-10/Gpr54 is a promising therapeutic target to abrogate bone resorption by Dusp18-mediated Src dephosphorylation.

SHC4 orchestrates ß-catenin pathway-mediated metastasis in triple-negative breast cancer by promoting Src kinase autophosphorylation.

Triple-negative breast cancer (TNBC) is highly aggressive and metastatic, and has the poorest prognosis among all breast cancer subtypes. Activated ß-catenin is enriched in TNBC and involved in Wnt signaling-independent metastasis. However, the underlying mechanisms of ß-catenin activation in TNBC remain unknown. Here, we found that SHC4 was upregulated in TNBC and high SHC4 expression was significantly correlated with poor outcomes. Overexpression of SHC4 promoted TNBC aggressiveness in vitro and facilitated TNBC metastasis in vivo. Mechanistically, SHC4 interacted with Src and maintained its autophosphorylated activation, which activated ß-catenin independent of Wnt signaling, and finally upregulated the transcription and expression of its downstream genes CD44 and MMP7. Furthermore, we determined that the PxPPxPxxxPxxP sequence on CH2 domain of SHC4 was critical for SHC4-Src binding and Src kinase activation. Overall, our results revealed the mechanism of ß-catenin activation independent of Wnt signaling in TNBC, which was driven by SHC4-induced Src autophosphorylation, suggesting that SHC4 might be a potential prognostic marker and therapeutic target in TNBC.

Profiling of drug resistance in Src kinase at scale uncovers a regulatory network coupling autoinhibition and catalytic domain dynamics.

Kinase inhibitors are effective cancer therapies, but resistance often limits clinical efficacy. Despite the cataloging of numerous resistance mutations, our understanding of kinase inhibitor resistance is still incomplete. Here, we comprehensively profiled the resistance of ∼3,500 Src tyrosine kinase mutants to four different ATP-competitive inhibitors. We found that ATP-competitive inhibitor resistance mutations are distributed throughout Src's catalytic domain. In addition to inhibitor contact residues, residues that participate in regulating Src's phosphotransferase activity were prone to the development of resistance. Unexpectedly, we found that a resistance-prone cluster of residues located on the top face of the N-terminal lobe of Src's catalytic domain contributes to autoinhibition by reducing catalytic domain dynamics, and mutations in this cluster led to resistance by lowering inhibitor affinity and promoting kinase hyperactivation. Together, our studies demonstrate how drug resistance profiling can be used to define potential resistance pathways and uncover new mechanisms of kinase regulation.

VE-cadherin junction dynamics in initial lymphatic vessels promotes lymph node metastasis.

The endothelial junction component vascular endothelial (VE)-cadherin governs junctional dynamics in the blood and lymphatic vasculature. Here, we explored how lymphatic junction stability is modulated by elevated VEGFA signaling to facilitate metastasis to sentinel lymph nodes. Zippering of VE-cadherin junctions was established in dermal initial lymphatic vessels after VEGFA injection and in tumor-proximal lymphatics in mice. Shape analysis of pan-cellular VE-cadherin fragments revealed that junctional zippering was accompanied by accumulation of small round-shaped VE-cadherin fragments in the lymphatic endothelium. In mice expressing a mutant VEGFR2 lacking the Y949 phosphosite (Vegfr2 Y949F/Y949F ) required for activation of Src family kinases, zippering of lymphatic junctions persisted, whereas accumulation of small VE-cadherin fragments was suppressed. Moreover, tumor cell entry into initial lymphatic vessels and subsequent metastatic spread to lymph nodes was reduced in mutant mice compared with WT, after challenge with B16F10 melanoma or EO771 breast cancer. We conclude that VEGFA mediates zippering of VE-cadherin junctions in initial lymphatics. Zippering is accompanied by increased VE-cadherin fragmentation through VEGFA-induced Src kinase activation, correlating with tumor dissemination to sentinel lymph nodes.

G protein­mediated EGFR transactivation is a common mechanism through which the CXCL12 receptors, CXCR4 and CXCR7, control human cancer cell migration.

C­X­C motif chemokine 12 (CXCL12) promotes metastasis of several tumors by affecting cell migration and invasion via its receptors, C­X­C chemokine receptor type (CXCR)4 and CXCR7. Current therapeutic approaches focus on the selective inactivation of either CXCR4 or CXCR7 in patients with cancer. Alternative strategies may emerge from the analysis of downstream events that mediate the migratory effects of CXCL12 in cancer cells. While CXCR4 activates cell signaling through both G proteins and arrestins, CXCR7 is believed to preferentially signal through arrestins. The present study analyzed the CXCL12­dependent chemotaxis of A549, C33A, DLD­1, MDA­MB­231 and PC­3 cells, in which either the activity of G proteins, EGFR or Src kinase was inhibited pharmacologically or the expression of arrestins was inhibited by RNA interference. The results demonstrated that CXCL12­induced migration of A549, C33A, DLD­1, MDA­MB­231 and PC­3 cells was attenuated by the Gαi/o­inhibitor pertussis toxin (PTX), but was unaffected by small interfering RNA­mediated gene silencing of ß­arrestin1/2. In particular, the sensitivity of DLD­1 migration to PTX was unexpected, as it is solely dependent on the non­classical chemokine receptor, CXCR7. Furthermore, chemotactic responses to CXCL12 were additionally prevented by inhibiting EGFR activity via AG1478 and Src kinase activity via Src inhibitor­1. In conclusion, the results of the present study suggest that G protein­ and Src­dependent transactivation of EGFR is a common mechanism through which CXCL12­bound CXCR4 and/or CXCR7 control cancer cell migration and metastasis. These findings highlight EGFR as a potential therapeutic target that interferes with CXCL12­induced cancer expansion.

A renaissance for YES in cancer.

Most of our understanding regarding the involvement of SRC-family tyrosine kinases in cancer has stemmed from studies focused on the prototypical SRC oncogene. However, emerging research has shed light on the important role of YES signaling in oncogenic transformation, tumor growth, metastatic progression, and resistance to various cancer therapies. Clinical evidence indicates that dysregulated expression or activity of YES is a frequent occurrence in human cancers and is associated with unfavorable outcomes. These findings provide a compelling rationale for specifically targeting YES in certain cancer subtypes. Here, we review the crucial role of YES in cancer and discuss the challenges associated with translating preclinical observations into effective YES-targeted therapies.

STAP-2 negatively regulates BCR-mediated B cell activation by recruiting tyrosine-protein kinase CSK to LYN.

Although signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN.

Integrated signaling and transcriptome analysis reveals Src family kinase individualities and novel pathways controlled by their constitutive activity.

The Src family kinases (SFKs) Lck and Lyn are crucial for lymphocyte development and function. Albeit tissue-restricted expression patterns the two kinases share common functions; the most pronounced one being the phosphorylation of ITAM motifs in the cytoplasmic tails of antigenic receptors. Lck is predominantly expressed in T lymphocytes; however, it can be ectopically found in B-1 cell subsets and numerous pathologies including acute and chronic B-cell leukemias. The exact impact of Lck on the B-cell signaling apparatus remains enigmatic and is followed by the long-lasting question of mechanisms granting selectivity among SFK members. In this work we sought to investigate the mechanistic basis of ectopic Lck function in B-cells and compare it to events elicited by the predominant B-cell SFK, Lyn. Our results reveal substrate promiscuity displayed by the two SFKs, which however, is buffered by their differential susceptibility toward regulatory mechanisms, revealing a so far unappreciated aspect of SFK member-specific fine-tuning. Furthermore, we show that Lck- and Lyn-generated signals suffice to induce transcriptome alterations, reminiscent of B-cell activation, in the absence of receptor/co-receptor engagement. Finally, our analyses revealed a yet unrecognized role of SFKs in tipping the balance of cellular stress responses, by promoting the onset of ER-phagy, an as yet completely uncharacterized process in B lymphocytes.

Activated Src kinase promotes cell cannibalism in Drosophila.

Src family kinases (SFKs) are evolutionarily conserved proteins acting downstream of receptors and regulating cellular processes including proliferation, adhesion, and migration. Elevated SFK expression and activity correlate with progression of a variety of cancers. Here, using the Drosophila melanogaster border cells as a model, we report that localized activation of a Src kinase promotes an unusual behavior: engulfment of one cell by another. By modulating Src expression and activity in the border cell cluster, we found that increased Src kinase activity, either by mutation or loss of a negative regulator, is sufficient to drive one cell to engulf another living cell. We elucidate a molecular mechanism that requires integrins, the kinases SHARK and FAK, and Rho family GTPases, but not the engulfment receptor Draper. We propose that cell cannibalism is a result of aberrant phagocytosis, where cells with dysregulated Src activity fail to differentiate between living and dead or self versus non-self, thus driving this malignant behavior.

Src tyrosine kinase promotes cardiac remodeling induced by chronic sympathetic activation.

Cardiac remodeling serves as the underlying pathological basis for numerous cardiovascular diseases and represents a pivotal stage for intervention. The excessive activation of ß-adrenergic receptors (ß-ARs) assumes a crucial role in cardiac remodeling. Nonetheless, the underlying molecular mechanisms governing ß-AR-induced cardiac remodeling remain largely unresolved. In the present study, we identified Src tyrosine kinase as a key player in the cardiac remodeling triggered by excessive ß-AR activation. Our findings demonstrated that Src mediates isoproterenol (ISO)-induced cardiac hypertrophy, fibrosis, and inflammation in vivo. Furthermore, Src facilitates ß-AR-mediated proliferation and transdifferentiation of cardiac fibroblasts, and hypertrophy and cardiomyocytes in vitro. Subsequent investigations have substantiated that Src mediates ß-AR induced the extracellular signal-regulated protein kinase (ERK1/2) signaling pathway activated by ß-AR. Our research presents compelling evidence that Src promotes ß-AR-induced cardiac remodeling in both in vivo and in vitro settings. It establishes the promoting effect of the ß-AR/Src/ERK signaling pathway on overall cardiac remodeling in cardiac fibroblasts and underscores the potential of Src as a therapeutic target for cardiac remodeling.

Using evolutionary constraint to define novel candidate driver genes in medulloblastoma.

Current knowledge of cancer genomics remains biased against noncoding mutations. To systematically search for regulatory noncoding mutations, we assessed mutations in conserved positions in the genome under the assumption that these are more likely to be functional than mutations in positions with low conservation. To this end, we use whole-genome sequencing data from the International Cancer Genome Consortium and combined it with evolutionary constraint inferred from 240 mammals, to identify genes enriched in noncoding constraint mutations (NCCMs), mutations likely to be regulatory in nature. We compare medulloblastoma (MB), which is malignant, to pilocytic astrocytoma (PA), a primarily benign tumor, and find highly different NCCM frequencies between the two, in agreement with the fact that malignant cancers tend to have more mutations. In PA, a high NCCM frequency only affects the BRAF locus, which is the most commonly mutated gene in PA. In contrast, in MB, >500 genes have high levels of NCCMs. Intriguingly, several loci with NCCMs in MB are associated with different ages of onset, such as the HOXB cluster in young MB patients. In adult patients, NCCMs occurred in, e.g., the WASF-2/AHDC1/FGR locus. One of these NCCMs led to increased expression of the SRC kinase FGR and augmented responsiveness of MB cells to dasatinib, a SRC kinase inhibitor. Our analysis thus points to different molecular pathways in different patient groups. These newly identified putative candidate driver mutations may aid in patient stratification in MB and could be valuable for future selection of personalized treatment options.

Whole-genome bisulfite sequencing identified the key role of the Src family tyrosine kinases and related genes in systemic lupus erythematosus.

BACKGROUND: As a multisystemic autoimmune illness, the basic mechanisms behind the pathophysiology of systemic lupus erythematosus (SLE) remain poorly understood. OBJECTIVE: We aimed to investigate the possible significance of SLE's DNA methylation and gain further insight into potential SLE-related biomarkers and therapeutic targets. METHODS: We used whole genome bisulfite sequencing (WGBS) method to analyze DNA methylation in 4 SLE patients and 4 healthy people. RESULTS: 702 differentially methylated regions (DMRs) were identified, and 480 DMR-associated genes were annotated. We found the majority of the DMR-associated elements were enriched in repeat and gene bodies. The top 10 hub genes identified were LCK, FYB, PTK2B, LYN, CTNNB1, MAPK1, GNAQ, PRKCA, ABL1, and CD247. Compared to the control group, LCK and PTK2B had considerably decreased levels of mRNA expression in the SLE group. Receiver operating characteristic (ROC) curve suggested that LCK and PTK2B may be potential candidate biomarkers to predict SLE. CONCLUSIONS: Our study improved comprehension of the DNA methylation patterns of SLE and identified potential biomarkers and therapeutic targets for SLE.

T-bet-expressing B cells contribute to the autoreactive plasma cell pool in Lyn-/- mice.

Systemic Lupus Erythematosus (SLE) is characterized by pathogenic autoantibodies against nucleic acid-containing antigens. Understanding which B-cell subsets give rise to these autoantibodies may reveal therapeutic approaches for SLE that spare protective responses. Mice lacking the tyrosine kinase Lyn, which limits B and myeloid cell activation, develop lupus-like autoimmune diseases characterized by increased autoreactive plasma cells (PCs). We used a fate-mapping strategy to determine the contribution of T-bet+ B cells, a subset thought to be pathogenic in lupus, to the accumulation of PCs and autoantibodies in Lyn-/- mice. Approximately, 50% of splenic PCs in Lyn-/- mice originated from T-bet+ cells, a significant increase compared to WT mice. In vitro, splenic PCs derived from T-bet+ B cells secreted both IgM and IgG anti-dsDNA antibodies. To determine the role of these cells in autoantibody production in vivo, we prevented T-bet+ B cells from differentiating into PCs or class switching in Lyn-/- mice. This resulted in a partial reduction in splenic PCs and anti-dsDNA IgM and complete abrogation of anti-dsDNA IgG. Thus, T-bet+ B cells make an important contribution to the autoreactive PC pool in Lyn-/- mice.

Molecular determinants of Hsp90 dependence of Src kinase revealed by deep mutational scanning.

Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and ß1-ß2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and ß1-ß2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions.

Comprehensive Analysis of Prognostic Value and Immune Infiltration of Src Family Kinases in Hepatocellular Carcinoma.

BACKGROUND: Src family kinases (SFKs) belong to the non-receptor protein tyrosine kinase family and are generally dysregulated in a variety of tumors. This study aimed to thoroughly investigate the mutation status, expression level, prognostic value and relationship with immune infiltration of SFKs in hepatocellular carcinoma (HCC). METHODS: TIMER2.0, UALCAN, cBioPortal, Gene Expression Profiling Interactive Analysis (GEPIA) and Kaplan-Meier Plotter were used to analyze the differential expression, genetic alteration, prognostic value and immune cell infiltration of SFKs in HCC patients. Furthermore, we used quantitative real-time PCR (qPCR) and western blot (WB) analysis to measure SFKs mRNA and protein expression in matching specimens of normal tissue and HCC. We analyzed the biological effects of FYN in Huh7 cells and subcutaneous xenograft tumor model. We also studied the biological effects of SRC on Huh7 cells. RESULTS: The mRNA expression levels of LYN, SRC and SRM were elevated in HCC tissues, whereas FYN was reduced. Approximately 10% genetic alterations rate of SFKs was observed in HCC. The mRNA levels of BLK, BRK, FRK, FYN, LCK, LYN, SRC, SRM and YES were correlated with clinical cancer stage. Elevated FYN mRNA levels in HCC were positively correlated with overall survival (OS), whereas SRC was negatively correlated with OS. All SFKs members in HCC were significantly associated with at least half of the six immune-infiltrating cells, including B cells, macrophages, dendritic cells, neutrophils, CD4+ T cells and CD8+ T cells. Furthermore, we confirmed that the protein expression level of FYN was decreased in patients with HCC and in a human hepatoma cell line. Overexpression of FYN suppressed Huh7 cell proliferation, migration, invasion, and tumorigenesis in xenograft nude mice. Knockdown of SRC inhibited Huh7 cell proliferation, migration and invasion. CONCLUSIONS: Dysregulated FYN and SRC expression in HCC is associated with poor prognosis and may be used as novel prognostic biomarkers in patients with HCC.

Constitutively active Lyn kinase causes a cutaneous small vessel vasculitis and liver fibrosis syndrome.

Neutrophilic inflammation is a hallmark of many monogenic autoinflammatory diseases; pathomechanisms that regulate extravasation of damaging immune cells into surrounding tissues are poorly understood. Here we identified three unrelated boys with perinatal-onset of neutrophilic cutaneous small vessel vasculitis and systemic inflammation. Two patients developed liver fibrosis in their first year of life. Next-generation sequencing identified two de novo truncating variants in the Src-family tyrosine kinase, LYN, p.Y508*, p.Q507* and a de novo missense variant, p.Y508F, that result in constitutive activation of Lyn kinase. Functional studies revealed increased expression of ICAM-1 on induced patient-derived endothelial cells (iECs) and of ß2-integrins on patient neutrophils that increase neutrophil adhesion and vascular transendothelial migration (TEM). Treatment with TNF inhibition improved systemic inflammation; and liver fibrosis resolved on treatment with the Src kinase inhibitor dasatinib. Our findings reveal a critical role for Lyn kinase in modulating inflammatory signals, regulating microvascular permeability and neutrophil recruitment, and in promoting hepatic fibrosis.

Activated Src kinases downstream of BCR-ABL and Flt3 induces proteasomal degradation of SHIP1 by phosphorylation of tyrosine 1021.

Within the various subtypes of ALL, patients with a BCR-ABL-positive background as well as with a genetic change in the KMT2A gene have by far the worst survival probabilities. Interestingly, both subtypes are characterized by highly activated tyrosine kinases. SHIP1 serves as an important negative regulator of the PI3K/AKT signaling pathway, which is often constitutively activated in ALL. The protein expression of SHIP1 is decreased in most T-ALL and in some subgroups of B-ALL. In this study, we analyzed the expression of SHIP1 protein in detail in the context of groups with aberrant activated tyrosine kinases, namely BCR-ABL (Ph+) and Flt3 (KMT2A translocations). We demonstrate that constitutively activated Src kinases downstream of BCR-ABL and receptor tyrosine kinases reduce the SHIP1 expression in a SHIP1-Y1021 phosphorylated-dependent manner with subsequent ubiquitin marked proteasomal degradation. Inhibition of BCR-ABL (Imatinib), Flt3 (Quizartinib) or Src-Kinase-Family (Saracatinib) leads to significant reconstitution of SHIP1 protein expression. These results further support a functional role of SHIP1 as tumor suppressor protein and could be the basis for the establishment of a targeted therapy form.

Activation loop phosphorylation tunes conformational dynamics underlying Pyk2 tyrosine kinase activation.

Pyk2 is a multidomain non-receptor tyrosine kinase that undergoes a multistage activation mechanism. Activation is instigated by conformational rearrangements relieving autoinhibitory FERM domain interactions. The kinase autophosphorylates a central linker residue to recruit Src kinase. Pyk2 and Src mutually phosphorylate activation loops to confer full activation. While the mechanisms of autoinhibition are established, the conformational dynamics associated with autophosphorylation and Src recruitment remain unclear. We employ hydrogen/deuterium exchange mass spectrometry and kinase activity profiling to map the conformational dynamics associated with substrate binding and Src-mediated activation loop phosphorylation. Nucleotide engagement stabilizes the autoinhibitory interface, while phosphorylation deprotects both FERM and kinase regulatory surfaces. Phosphorylation organizes active site motifs linking catalytic loop with activation segment. Dynamics of the activation segment anchor propagate to EF/G helices to prevent reversion of the autoinhibitory FERM interaction. We employ targeted mutagenesis to dissect how phosphorylation-induced conformational rearrangements elevate kinase activity above the basal autophosphorylation rate.

B-lymphoid tyrosine kinase-mediated FAM83A phosphorylation elevates pancreatic tumorigenesis through interacting with ß-catenin.

Abnormal activation of Wnt/ß-catenin-mediated transcription is closely associated with the malignancy of pancreatic cancer. Family with sequence similarity 83 member A (FAM83A) was shown recently to have oncogenic effects in a variety of cancer types, but the biological roles and molecular mechanisms of FAM83A in pancreatic cancer need further investigation. Here, we newly discovered that FAM83A binds directly to ß-catenin and inhibits the assembly of the cytoplasmic destruction complex thus inhibiting the subsequent phosphorylation and degradation. FAM83A is mainly phosphorylated by the SRC non-receptor kinase family member BLK (B-lymphoid tyrosine kinase) at tyrosine 138 residue within the DUF1669 domain that mediates the FAM83A-ß-catenin interaction. Moreover, FAM83A tyrosine 138 phosphorylation enhances oncogenic Wnt/ß-catenin-mediated transcription through promoting ß-catenin-TCF4 interaction and showed an elevated nucleus translocation, which inhibits the recruitment of histone deacetylases by TCF4. We also showed that FAM83A is a direct downstream target of Wnt/ß-catenin signaling and correlates with the levels of Wnt target genes in human clinical pancreatic cancer tissues. Notably, the inhibitory peptides that target the FAM83A-ß-catenin interaction significantly suppressed pancreatic cancer growth and metastasis in vitro and in vivo. Our results revealed that blocking the FAM83A cascade signaling defines a therapeutic target in human pancreatic cancer.