HIV-1 Nef interaction influences the ATP-binding site of the Src-family kinase, Hck

Background: Nef is an HIV-1 accessory protein essential for viral replication and AIDS progression. Nef interacts with a multitude of host cell signaling partners, including members of the Src kinase family. Nef preferentially activates Hck, a Src-family kinase (SFK) strongly expressed in macrophages and other HIV target cells, by binding to its regulatory SH3 domain. Recently, we identified a series of kinase inhibitors that preferentially inhibit Hck in the presence of Nef. These compounds also block Nef-dependent HIV replication, validating the Nef-SFK signaling pathway as an antiretroviral drug target. Our findings also suggested that by binding to the Hck SH3 domain, Nef indirectly affects the conformation of the kinase active site to favor inhibitor association. Results: To test this hypothesis, we engineered a "gatekeeper" mutant of Hck with enhanced sensitivity to the pyrazolopyrimidine tyrosine kinase inhibitor, NaPP1. We also modified the RT loop of the Hck SH3 domain to enhance interaction of the kinase with Nef. This modification stabilized Nef:Hck interaction in solution-based kinase assays, as a way to mimic the more stable association that likely occurs at cellular membranes. Introduction of the modified RT loop rendered Hck remarkably more sensitive to activation by Nef, and led to a significant decrease in the K mssssfor ATP as well as enhanced inhibitor potency. Conclusions: These observations suggest that stable interaction with Nef may induce Src-family kinase active site conformations amenable to selective inhibitor targeting. © 2012 Pene-Dumitrescu et al; licensee BioMed Central Ltd

Inhibition of N1-Src kinase by a specific SH3 peptide ligand reveals a role for N1-Src in neurite elongation by L1-CAM

In the mammalian brain the ubiquitous tyrosine kinase, C-Src, undergoes splicing to insert short sequences in the SH3 domain to yield N1- and N2-Src. We and others have previously shown that the N-Srcs have altered substrate specificity and kinase activity compared to C-Src. However, the exact functions of the N-Srcs are unknown and it is likely that N-Src signalling events have been misattributed to C-Src because they cannot be distinguished by conventional Src inhibitors that target the kinase domain. By screening a peptide phage display library, we discovered a novel ligand (PDN1) that targets the unique SH3 domain of N1-Src and inhibits N1-Src in cells. In cultured neurons, PDN1 fused to a fluorescent protein inhibited neurite outgrowth, an effect that was mimicked by shRNA targeting the N1-Src microexon. PDN1 also inhibited L1-CAM-dependent neurite elongation in cerebellar granule neurons, a pathway previously shown to be disrupted in Src(−/−) mice. PDN1 therefore represents a novel tool for distinguishing the functions of N1-Src and C-Src in neurons and is a starting point for the development of a small molecule inhibitor of N1-Src

SRC signaling in cancer and tumor microenvironment

PubMed: 33123993Pioneering experiments performed by Harold Varmus and Mike Bishop in 1976 led to one of the most influential discoveries in cancer research and identified the first cancer-causing oncogene called Src. Later experimental and clinical evidence suggested that Src kinase plays a significant role in promoting tumor growth and progression and its activity is associated with poor patient survival. Thus, several Src inhibitors were developed and approved by FDA for treatment of cancer patients. Tumor microenvironment (TME) is a highly complex and dynamic milieu where significant cross-talk occurs between cancer cells and TME components, which consist of tumor-associated macrophages, fibroblasts, and other immune and vascular cells. Growth factors and chemokines activate multiple signaling cascades in TME and induce multiple kinases and pathways, including Src, leading to tumor growth, invasion/metastasis, angiogenesis, drug resistance, and progression. Here, we will systemically evaluate recent findings regarding regulation of Src and significance of targeting Src in cancer therapy. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2021