Protein kinase affinity reagents based on a 5-aminoindazole scaffold.

Affinity reagents that target protein kinases are powerful tools for signal transduction research. Here, we describe a general set of kinase ligands based on a 5-aminoindazole scaffold. This scaffold can readily be derivatized with diverse binding elements and immobilized analogs allow selective enrichment of protein kinases from complex mixtures.

Biochemical and pharmacological profiling of the pro-survival protein Bcl-xL.

Bcl-2 family proteins are key mediators of programmed cell death. Over-expression of anti-apoptotic Bcl-2 family members (Bcl-2, Bcl-xL, and Mcl-1) has been associated with tumor progression and chemotherapeutic resistance. Pharmacological agents that neutralize the functions of anti-apoptotic Bcl-2 family proteins have emerged as a promising new class of anti-cancer agents. Biochemical analyses have demonstrated that small molecule inhibitors and some pro-apoptotic proteins exhibit distinct binding preferences for anti-apoptotic proteins. While numerous structures of anti-apoptotic proteins bound to ligands have been reported, the source of this selectivity is still unclear. Here, we present a systematic analysis of a series of Bcl-xL variants that contain mutations within the hydrophobic ligand-binding cleft. The ability of these Bcl-xL mutants to interact with both small molecule inhibitors and BH3 peptides was determined. These studies provide information on the contributions of specific residues to small molecule inhibitor binding and shed light on the ligand selectivity of these therapeutically important proteins.

NEDD9 stimulated MMP9 secretion is required for invadopodia formation in oral squamous cell carcinoma.

Neural precursor cell expressed developmentally downregulated 9 (NEDD9) is a component of the metastatic signatures of melanoma, breast cancer, glioblastoma, lung cancer and head and neck squamous cell carcinoma (HNSCC). Here we tested the efficacy of NEDD9's domains in stimulating matrix metalloproteinase (MMP) secretion and invadopodia formation in cells stably expressing various NEDD9 mutants. Replacement of the 13 YxxP motif substrate domain (SD) tyrosines and the C-terminal Y629 with phenylalanines (F14NEDD9) eliminated tyrosine phosphorylation, MMP9 secretion and loss of invadopodia formation. Mutation of the N-terminal SH3 domain Y12 to glutamic acid (Y12ENEDD9) or phenylalanine (Y12FNEDD9) reduced MMP9 secretion and inhibited invadopodia formation. SH3 domain deletion (∆SH3NEDD9) resulted in the loss of MMP9 secretion and a lack of invadopodia formation. The SH3-SD domain (SSNEDD9) construct exhibited tyrosine phosphorylation and stimulated MMP9 secretion, as did ∆CTNEDD9 which lacked the C-terminus (∆C-terminal; ∆CT). E13NEDD9 expression blocked MMP9 secretion and invadopodia formation. MICAL1 (molecule interacting with Cas-L1) silencing with a short hairpin RNA reduced MMP9 secretion, vimentin and E-cadherin levels while increasing N-cadherin and Rab6 levels, consistent with reduced invasive behavior. These findings indicate that NEDD9 SD phosphorylation and SH3 domain interactions are necessary for increasing MMP9 secretion and invadopodia formation.

Affinity reagents that target a specific inactive form of protein kinases.

A number of small-molecule inhibitors have been developed that target the catalytic domains of protein kinases that are not in an active conformation. An inactive form that has been observed in several kinases is the DFG-out conformation. This conformation is characterized by an almost 180 degrees rotation of the conserved Asp-Phe-Gly (DFG) motif in the ATP-binding cleft relative to the active form. However, the sequence and structural determinants that allow a kinase to stably adopt the DFG-out conformation are not known. Here, we characterize a series of inhibitors based on a general pharmacophore for this inactive form. We demonstrate that modified versions of these inhibitors can be used to study the thermodynamics and kinetics of ligand binding to DFG-out-adopting kinases and for enriching these kinases from complex protein mixtures.