The other authors declare no competing interests

The other authors declare no competing interests. Notes Published: March 23, 2018 Footnotes Supplemental Information includes Transparent Methods, four Loviride figures, and five tables and can be found with this article online at Supplemental Information Document S1. presence of full-length c-Src, v-Src, or c-Abl tyrosine kinases. Loviride Following pulldown, immunoblotting was performed to assess tyrosine phosphorylation using phos-Tyr, 4G10 antibody. (F) TIMP-2 constructs were transiently expressed in SYF and SYF?+ c-Src cells, pulled down from 10X concentrated CM Loviride and immunoblotted to determine TIMP-2 tyrosine phosphorylation. See also Figure?S1. To determine the tyrosine kinase responsible for this phosphorylation, we hypothesized that phosphorylation occurs at key sequence motifs surrounding the targeted residues. Amino acid isoleucine (I) at the C 1 position of Y62 and Y90 (kinase assay using recombinant unphosphorylated TIMP-2-His6 (rTIMP-2-His6; Figure?1E). Following pulldown Ni-NTA, we found that c-Src and more intensely v-Src phosphorylate rTIMP-2-His6 (Figure?1E). v-Src is known to represent the hyperactive oncogenic version of c-Src because of its truncated inhibitory C-terminal regulatory phosphorylation site (Y527; Roskoski, 2004). We quantitatively measured and confirmed the hyperactivity of v-Src used Rabbit Polyclonal to CSTL1 here (Figure?S1B). We also found that c-Abl tyrosine kinase did not phosphorylate TIMP-2 (Figure?1E), and in control experiments using heat shock protein 90 alpha (Hsp90) as substrate we demonstrate discriminatory phosphorylation by Src kinase but not for c-Abl, confirming our previous work (Beebe et?al., 2013, Dunn et?al., 2015, Mollapour et?al., 2010; Figures 1E and S1C). To test the phosphorylation of TIMP-2, we transiently expressed WT TIMP-2-His6 and mutants in a triple kinase knockout (c-Src, Yes, and Fyn) mouse embryonic fibroblast cell line SYF and in cells with wild-type c-Src reintroduced (SYF?+ c-Src; Figure?1F). Pulldown experiments confirmed tyrosine phosphorylation of WT TIMP-2 in the SYF?+ c-Src but not in the parental SYF cell CM (Figure?1F). Since phosphorylation at Y62F, Y90F, and Y165F is reduced, and TF lacks phosphorylation in the SYF?+ c-Src CM, the overall findings suggest that c-Src targets all three tyrosine residues. Secreted c-Src Phosphorylates TIMP-2 in the Extracellular Space TIMP-2 contains an amino-terminal signal sequence that directs the newly synthesized protein to the ER, followed by secretion via the ER/Golgi pathway (Benham, 2012; Figure?S1A). c-Src is a cytosolic kinase known to phosphorylate substrates at sites localized within the cell. To elucidate the cellular compartment where c-Src phosphorylates TIMP-2, we first assessed c-Src secretion from cells. CM was collected from mammalian cell lines and following normalization to total cellular protein levels, samples were analyzed by immunoblotting (Figures S2A and S2B). c-Src protein was present at varying levels in the extracts and CM from all cell lines tested (Figures S2A and S2B). Absence of cytosolic GAPDH in the CM also verifies lack of cytoplasmic fractions as a result of cell injury. Next, we asked if Loviride TIMP-2 could have been phosphorylated before secretion. HEK293H cells were transiently transfected with WT TIMP-2-His6, followed by 12-hr serum starvation and treatment with brefeldin A, an inhibitor that blocks conventional secretion by disrupting protein transport from ER to Golgi (Figure?2A). As expected, brefeldin A hindered TIMP-2 secretion but not the secretion of c-Src (Figure?2A). Tyrosine phosphorylation was also abolished in TIMP-2 isolated from cell extracts (Figure?2A). These data indicate that tyrosine phosphorylation occurs following TIMP-2 transport to Golgi or extracellularly. As c-Src secretion remains unaffected, we hypothesized that the phosphorylation occurs following secretion. To test this, we serum starved HT1080 cells for 18?hr and then supplemented the CM with rTIMP-2-His6 (Figure?2B). We detected both TIMP-2 and c-Src in the CM, 2 and 8?hr post treatment (Figure?2B). Notably, the exogenously added rTIMP-2-His6 is also detected in cell extracts, with protein levels increasing over time, indicating that a certain amount of free rTIMP-2-His6 becomes cell associated (Figure?2B). It is, therefore, not surprising to find that both cell-associated and free TIMP-2 are tyrosine phosphorylated (Figure?2B). To strengthen our findings on extracellular phosphorylation, we assessed the effects of anti-c-Src antibodies as possible inhibitors of TIMP-2 tyrosine phosphorylation (Table S1. [Information of antibodies used]). We pre-treated Loviride HT1080 cultures with an anti-c-Src antibody (mAb1) or rabbit IgG control before the addition of rTIMP-2-His6. As predicted, TIMP-2 tyrosine phosphorylation was impaired in both cell extracts.