The Far Side of Resistance to RAS Inhibitors.

In this issue, four articles highlight the critical role of nongenetic mechanisms and cell plasticity in mediating resistance to different classes of RAS inhibitors in pancreatic ductal adenocarcinoma and non-small cell lung cancer. See related article by Benitz et al., p.

Phosphopeptide binding to the N-SH2 domain of tyrosine phosphatase SHP2 correlates with the unzipping of its central β-sheet.

SHP2 is a tyrosine phosphatase that plays a regulatory role in multiple intracellular signaling cascades and is known to be oncogenic in certain contexts. In the absence of effectors, SHP2 adopts an autoinhibited conformation with its N-SH2 domain blocking the active site. Given the key role of N-SH2 in regulating SHP2, this domain has been extensively studied, often by X-ray crystallography.

NMR Methods to Study the Dynamics of SH2 Domain-Phosphopeptide Complexes.

Nuclear magnetic resonance (NMR) spectroscopy is the method of choice for studying the dynamics of biological macromolecules in solution. By exploiting the intricate interplay between the effects of protein motion (both overall rotational diffusion and internal mobility) and nuclear spin relaxation, NMR allows molecular motion to be probed at atomic resolution over a wide range of timescales, including picosecond (bond vibrations and methyl-group rotations), nanosecond (loop motions and rotational diffusion), and microsecond-millisecond (ligand binding, allostery). In this chapter, we describe different NMR pulse schemes (R, R, heteronuclear NOE, and CPMG relaxation dispersion) to characterize the dynamics of SH2 domains.

Methods for Structure Determination of SH2 Domain-Phosphopeptide Complexes by NMR.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique to solve the structure of biomolecular complexes at atomic resolution in solution. Small proteins such as Src-homology 2 (SH2) domains have fast tumbling rates and long-lived NMR signals, making them particularly suited to be studied by standard NMR methods. SH2 domains are modular proteins whose function is the recognition of sequences containing phosphotyrosines.

Experiment-guided molecular simulations define a heterogeneous structural ensemble for the tandem SH2 domains.

SHP2 plays an important role in regulating cellular processes, and its pathogenic mutations cause developmental disorders and are linked to cancer. SHP2 is a multidomain protein, comprising two SH2 domains arranged in tandem, a catalytic PTP domain, and a disordered C-terminal tail. SHP2 is activated upon binding two linked phosphopeptides to its SH2 domains, and the peptide orientation and spacing between binding sites are critical for enzymatic activation.

Molecular Characterization of Acquired Resistance to KRASG12C-EGFR Inhibition in Colorectal Cancer.

Unlabelled: With the combination of KRASG12C and EGFR inhibitors, KRAS is becoming a druggable target in colorectal cancer. However, secondary resistance limits its efficacy. Using cell lines, patient-derived xenografts, and patient samples, we detected a heterogeneous pattern of putative resistance alterations expected primarily to prevent inhibition of ERK signaling by drugs at progression.

Anatomic position determines oncogenic specificity in melanoma.

Oncogenic alterations to DNA are not transforming in all cellular contexts. This may be due to pre-existing transcriptional programmes in the cell of origin. Here we define anatomic position as a major determinant of why cells respond to specific oncogenes.

Recruitment of phospholipase Cγ1 to the non-structural membrane protein pK15 of Kaposi Sarcoma-associated herpesvirus promotes its Src-dependent phosphorylation.

Kaposi Sarcoma-associated herpesvirus (KSHV) causes three human malignancies, Kaposi Sarcoma (KS), Primary Effusion Lymphoma (PEL) and the plasma cell variant of multicentric Castleman's Disease (MCD), as well as an inflammatory cytokine syndrome (KICS). Its non-structural membrane protein, pK15, is among a limited set of viral proteins expressed in KSHV-infected KS tumor cells. Following its phosphorylation by Src family tyrosine kinases, pK15 recruits phospholipase C gamma 1 (PLCγ1) to activate downstream signaling cascades such as the MEK/ERK, NFkB and PI3K pathway, and thereby contributes to the increased proliferation and migration as well as the spindle cell morphology of KSHV-infected endothelial cells.

Phosphotyrosine couples peptide binding and SHP2 activation via a dynamic allosteric network.

SHP2 is a ubiquitous protein tyrosine phosphatase, whose activity is regulated by phosphotyrosine (pY)-containing peptides generated in response to extracellular stimuli. Its crystal structure reveals a closed, auto-inhibited conformation in which the N-terminal Src homology 2 (N-SH2) domain occludes the catalytic site of the phosphatase (PTP) domain. High-affinity mono-phosphorylated peptides promote catalytic activity by binding to N-SH2 and disrupting the interaction with the PTP.

Specificity and regulation of phosphotyrosine signaling through SH2 domains.

Phosphotyrosine (pY) signaling is instrumental to numerous cellular processes. pY recognition occurs through specialized protein modules, among which the Src-homology 2 (SH2) domain is the most common. SH2 domains are small protein modules with an invariant fold, and are present in more than a hundred proteins with different function.

H, C, N chemical shift assignments of SHP2 SH2 domains in complex with PD-1 immune-tyrosine motifs.

Inhibition of immune checkpoint receptor Programmed Death-1 (PD-1) via monoclonal antibodies is an established anticancer immunotherapeutic approach. This treatment has been largely successful; however, its high cost demands equally effective, more affordable alternatives. To date, the development of drugs targeting downstream players in the PD-1-dependent signaling pathway has been hampered by our poor understanding of the molecular details of the intermolecular interactions involved in the pathway.