Src Kinase Is Biphosphorylated at Y416/Y527 and Activates the CUB-Domain Containing Protein 1/Protein Kinase C δ Pathway in a Subset of Triple-Negative Breast Cancers

Abstract

Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study, we investigated the activation of Src family of cytoplasmic tyrosine kinases (SFKs) and two SFK substratesdCUB-domain containing protein 1 (CDCP1) and protein kinase C d (PKCd)din 56 formalin-fixed, paraffin-embedded (FFPE) TNBCs. Expression of SFK phosphorylated at Y416 (SFK_pY416 þ ) in tumor cells was strongly associated with phosphorylation of CDCP1 and PKCd (CDCP1_ pY743 þ and PKCd_pY311 þ ), as assessed by immunohistochemistry, indicating increased SFK activity in situ. To enable biochemical analysis, protein extraction from FFPE tissue was optimized. Cleaved CDCP1 isoform (70 kDa) was expressed to a varying degree in all samples but only phosphorylated in TNBC tumor cells that expressed SFK_pY416. Interestingly, active SFK was found to be biphosphorylated (SFK_pY416 þ /pY527 þ ). Biphosphorylated active SFK was observed more frequently in forkhead box protein A1 (FOXA1) À TNBCs. In addition, in SFK_pY416 À samples, FOXA1 þ TNBC tended to be SFK_pY527 þ (classic inactive SFK), and FOXA1 À TNBC tended to be SFK_pY527 À (SFK poised for activation). Strong SFK_pY416 staining was also observed in tumor-infiltrating lymphocytes in a subset of TNBCs with high tumor-infiltrating lymphocyte content. This report will facilitate protein biochemical analysis of FFPE tumor samples and justifies the development of therapies targeting the SFK/ CDCP1/PKCd pathway for TNBC treatment.

A role of Lck annular lipids in the steady upkeep of active Lck in T cells

“…1A ). This is consistent with in vitro or in cellulo data that Lck (Hui and Vale, 2014) and Src (Nelson et al, 2020; Sun et al, 1998) can phosphorylate the C-terminal regulatory tyrosine. For evident steric constraints, double phosphorylated Src cannot close (Sun et al, 1998), consistent with Lck ADP featuring in vitro kinase activity similar to Lck A (Hui and Vale, 2014; Nika et al, 2010).…”

Src-family Protein Tyrosine Kinases: A promising target for treating Cardiovascular Diseases

“…The intracellular location is dynamic, such as the enrichment of activated Fyn in multivesicular bodies led to a defect in cell differentiation in a neuroblastoma cell line 90 . Next, the study on the pathological mechanism of SFKs in the above-mentioned diseases is not in-depth enough, SFKs can phosphorylate multiple sites of relative proteins in mammalian cells 91 , or the presence of bi-phosphorylated SFKs was found in triple-negative breast cancer model 92 . The phosphorylation sites of key upstream targets and receptor proteins related to CVDs need to be more accurate for a deep research from many aspects and levels.…”

An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

“…Large-scale genomic sequencing projects indicate that gene amplification and activating mutations in c-Src do not play a significant role in human tumor biology (Bailey et al, 2018; Curtis et al, 2012). In fact, paradoxical high levels of phosphorylated c-Src in both activation and c-terminal segments are found in aggressive cancer types such TNBC (Nelson et al, 2020) and NSCLC (unpublished). We have data supporting these findings where a previously phosphorylated c-Src protein (90-120 min) is active and able to phosphorylate an intact susbtrate surrogate with faster kinetics than the non-phosphorylated protein (Fig.…”