Redox Regulation of Brain Selective Kinases BRSK1/2: Implications for Dynamic Control of the Eukaryotic AMPK family through Cys-based mechanisms.

In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications (PTMs), including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related Brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intramolecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-Loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

Targeting the WASF3 complex to suppress metastasis.

Wiskott-Aldrich syndrome protein family members (WASF) regulate the dynamics of the actin cytoskeleton, which plays an instrumental role in cancer metastasis and invasion. WASF1/2/3 forms a hetero-pentameric complex with CYFIP1/2, NCKAP1/1 L, Abi1/2/3 and BRK1 called the WASF Regulatory Complex (WRC), which cooperatively regulates actin nucleation by WASF1/2/3. Activation of the WRC enables actin networking and provides the mechanical force required for the formation of lamellipodia and invadopodia. Although the WRC drives cell motility essential for several routine physiological functions, its aberrant deployment is observed in cancer metastasis and invasion. WASF3 expression is correlated with metastatic potential in several cancers and inversely correlates with overall progression-free survival. Therefore, disruption of the WRC may serve as a novel strategy for targeting metastasis. Given the complexity involved in the formation of the WRC which is largely comprised of large protein-protein interfaces, there are currently no inhibitors for WASF3. However, several constrained peptide mimics of the various protein-protein interaction interfaces within the WRC were found to successfully disrupt WASF3-mediated migration and invasion. This review explores the role of the WASF3 WRC in driving metastasis and how it may be selectively targeted for suppression of metastasis.

In Silico Optimized Stapled Peptides Targeting WASF3 in Breast Cancer.

Wiskott-Aldrich Syndrome Protein Family (WASF) members regulate actin cytoskeletal dynamics, and WASF3 is directly associated with breast cancer metastasis and invasion. WASF3 forms a heteropentameric complex with CYFIP, NCKAP, ABI, and BRK1, called the WASF Regulatory Complex (WRC), which cooperatively regulates actin nucleation by WASF3. Since aberrant deployment of the WRC is observed in cancer metastasis and invasion, its disruption provides a novel avenue for targeting motility in breast cancer cells. Here, we report the development of a second generation WASF3 mimetic peptide, WAHMIS-2, which was designed using a combination of structure-guided design, homology modeling, and in silico optimization to disrupt binding of WASF3 to the WRC. WAHMIS-2 was found to permeate cells and inhibit cell motility, invasion, and MMP9 expression with greater potency than its predecessor, WAHM1. Targeted disruption of WASF3 from the WRC may serve as a useful strategy for suppression of breast cancer metastasis.

Targeted disruption of PKC from AKAP signaling complexes.

Protein Kinase C (PKC) is a member of the AGC subfamily of kinases and regulates a wide array of signaling pathways and physiological processes. Protein-protein interactions involving PKC and its scaffolding partners dictate the spatiotemporal dynamics of PKC activity, including its access to activating second messenger molecules and potential substrates. While the A Kinase Anchoring Protein (AKAP) family of scaffold proteins universally bind PKA, several were also found to scaffold PKC, thereby serving to tune its catalytic output. Targeting these scaffolding interactions can further shed light on the effect of subcellular compartmentalization on PKC signaling. Here we report the development of two hydrocarbon stapled peptides, CSTAD5 and CSTAD6, that are cell permeable and bind PKC to disrupt PKC-gravin complex formation in cells. Both constrained peptides downregulate PMA-induced cytoskeletal remodeling that is mediated by the PKC-gravin complex as measured by cell rounding. Further, these peptides downregulate PKC substrate phosphorylation and cell motility. To the best of our knowledge, no PKC-selective AKAP disruptors have previously been reported and thus CSTAD5 and CSTAD6 are novel disruptors of PKC scaffolding by AKAPs and may serve as powerful tools for dissecting AKAP-localized PKC signaling.

A Stapled Peptide Mimic of the Pseudosubstrate Inhibitor PKI Inhibits Protein Kinase A.

Kinases regulate multiple and diverse signaling pathways and misregulation is implicated in a multitude of diseases. Although significant efforts have been put forth to develop kinase-specific inhibitors, specificity remains a challenge. As an alternative to catalytic inhibition, allosteric inhibitors can target areas on the surface of an enzyme, thereby providing additional target diversity. Using cAMP-dependent protein kinase A (PKA) as a model system, we sought to develop a hydrocarbon-stapled peptide targeting the pseudosubstrate domain of the kinase. A library of peptides was designed from a Protein Kinase Inhibitor (PKI), a naturally encoded protein that serves as a pseudosubstrate inhibitor for PKA. The binding properties of these peptide analogs were characterized by fluorescence polarization and surface plasmon resonance, and two compounds were identified with KD values in the 500-600 pM range. In kinase activity assays, both compounds demonstrated inhibition with 25-35 nM IC50 values. They were also found to permeate cells and localize within the cytoplasm and inhibited PKA activity within the cellular environment. To the best of our knowledge, these stapled peptide inhibitors represent some of the highest affinity binders reported to date for hydrocarbon stapled peptides.