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Phosphosecretome profiling and kinase-substrate interaction screening reveal key extracellular c-Src-regulated pathways

ABSTRACT: Secreted protein kinases are responsible for the phosphorylation of a vast number of extracellular phosphoproteins. The tyrosine kinase c-Src is secreted through the vesicular trafficking pathway, however, mechanistic insights into extracellular substrate specificity and potential therapeutic opportunities in cancer remain unexplored. Here, we show that the SH4/Unique region of c-Src is required for its secretion. Limited proteolysis-mass spectrometry together with mutagenesis further confirmed predictions of in-silico molecular docking of active c-Src structure with its bona fide substrate TIMP2, that the Src homology 3 (SH3) domain is critical for kinase interaction with the P31xxP34 motif in the amino domain of TIMP2. Interestingly, comprehensive unbiased phosphoproteomic studies revealed an enrichment of PxxP containing extracellular substrates as potential targets of secreted c-Src. Cell-based studies using custom anti-c-Src antibodies against the c-Src SH3-TIMP2 interacting interface revealed that blocking secreted Src not only disrupts kinase-substrate interaction but also inhibits prostate cancer cell proliferation. Together, our data provide insights into the intricate role of secreted c-Src in extracellular phosphorylation.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Cell Culture, Fibroblast

SUBMITTER: Sarah Backe

LAB HEAD: Dimitra Bourboulia

PROVIDER: PXD036069 | Pride | 2023-05-30

REPOSITORIES: pride

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Project description:Ovarian estrogen (E2) and progesterone (P4) are indispensable for embryo-implantation and endometrial stromal decidualization; however, the molecular mechanisms that underpin these reproductive processes are unclear. Steroid receptor coregulator-2 (SRC-2) belongs to the multifunctional SRC/p160 family which also includes SRC-1 and SRC-3. Sharing strong sequence homology, all three SRCs exert diverse regulatory effects by modulating the transcriptional potency of nuclear receptor family members, including the estrogen and progesterone receptor (ER and PR respectively). Importantly, absence of SRC-2 in PR positive cells in the epithelial, stromal, and myometrial compartments of the murine uterus results in a striking infertility defect. This reproductive phenotype highlights a key role for SRC-2 in uterine function which is not shared with other coregulators. Intriguingly, abrogation of uterine SRC-2 does not block embryo apposition or attachment to the apical surface of luminal epithelial cells of the endometrium but rather prevents P4-dependent local decidualization of the sub-epithelial stroma. Remarkably, epithelial-specific ablation of SRC-2 in the murine uterus does not compromise endometrial functionality, again underscoring the unique importance of stromal derived SRC-2 in uterine function. The stromal decidualization defect resulting from SRC-2 ablation is reflected at the molecular level by a marked attenuation in P4 responsive target genes known to be critical for P4 dependent decidualization (i.e. ERBB receptor feedback inhibitor 1, Follistatin and Fkbp5). Conversely, the induction of E2 or P4 target genes involved in embryo implantation (i.e. leukemia inhibitory factor (LIF) and Indian hedgehog (Ihh) respectively) is not affected by SRC-2’s absence. As with mouse studies, decidualization of primary human stromal cells (HESCs) in culture is blocked by SRC-2 knockdown; however, HESC decidualization is unaffected by knockdown of SRC-1 or SRC-3. As a consequence of SRC-2 knockdown, molecular studies disclose a striking decrease in the induction of a subset of P4 target genes (i.e. WNT4 and FKBP5) which are essential for the stromal-epithelioid transformation step, the cellular hallmark of endometrial decidualization. Collectively, these studies not only showcase the evolutionary importance of SRC-2 in endometrial biology but also suggest that deregulation of this coregulator may underpin a spectrum of hormone-dependent uterine pathologies such as endometriosis and endometrial cancer. Microarray analysis was performed on mouse uteri using eighteen SRC-2flox/flox (SRC-2f/f) and eighteen PRCre/+ SRC-2flox/flox (SRC-2d/d) mice. Mice were ovariectomized at 6 weeks and after 2 weeks mice were either treated with sesame oil (vehicle) or 1 mg of P4. RNA from three mice per genotype per treatment were pooled and assigned as one sample (three samples per genotype per treatment). multiple group comparison

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	MM1072_peptides.xlsx	Xlsx

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Phosphosecretome profiling and kinase-substrate interaction screening reveal key extracellular c-Src-regulated pathways

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