Andrew Kaplan

Distinct Phosphotyrosine-dependent Functions of the ShcA Adaptor Protein Are Required for Transforming Growth Factor β (TGFβ)-induced Breast Cancer Cell Migration, Invasion, and Metastasis

Background: ShcA integrates TGFβ and ErbB2 signaling in breast cancer. Results: Distinct motifs within ShcA facilitate TGFβ-induced effects in ErbB2-expressing cells. Conclusion: ShcA transduces distinct signals via Grb2 downstream of Tyr313 and Crk adaptors downstream of Tyr239/Tyr240, and all three residues are required for metastasis. Significance: ShcA is essential for the tumorigenesis and metastasis of ErbB2-expressing breast tumors with active TGFβ signaling. The ErbB2 and TGFβ signaling pathways cooperate to promote the migratory, invasive, and metastatic behavior of breast cancer cells. We previously demonstrated that ShcA is necessary for these synergistic interactions. Through a structure/function approach, we now show that the phosphotyrosine-binding, but not the Src homology 2, domain of ShcA is required for TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. We further demonstrate that the tyrosine phosphorylation sites within ShcA (Tyr239/Tyr240 and Tyr313) transduce distinct and non-redundant signals that promote these TGFβ-mediated effects. We demonstrate that Grb2 is required specifically downstream of Tyr313, whereas the Tyr239/Tyr240 phosphorylation sites require the Crk adaptor proteins to augment TGFβ-induced migration and invasion. Furthermore, ShcA Tyr313 phosphorylation enhances tumor cell survival, and ShcA Tyr239/Tyr240 signaling promotes endothelial cell recruitment into ErbB2-expressing breast tumors in vivo, whereas all three ShcA tyrosine residues are required for efficient breast cancer metastasis to the lungs. Our data uncover a novel ShcA-dependent signaling axis downstream of TGFβ and ErbB2 that requires both the Grb2 and Crk adaptor proteins to increase the migratory and invasive properties of breast cancer cells. In addition, signaling downstream of specific ShcA tyrosine residues facilitates the survival, vascularization, and metastatic spread of breast tumors.

Hsc70 chaperone activity underlies Trio GEF function in axon growth and guidance induced by netrin-1

Hsc70 chaperone activity is required for Rac1 activation by Trio and this function underlies netrin-1/DCC-dependent axon outgrowth and guidance.

Extrinsic and intrinsic regulation of axon regeneration at a crossroads.

Repair of the injured spinal cord is a major challenge in medicine. The limited intrinsic regenerative response mounted by adult central nervous system (CNS) neurons is further hampered by astrogliosis, myelin debris and scar tissue that characterize the damaged CNS. Improved axon regeneration and recovery can be elicited by targeting extrinsic factors as well as by boosting neuron-intrinsic growth regulators. Our knowledge of the molecular basis of intrinsic and extrinsic regulators of regeneration has expanded rapidly, resulting in promising new targets to promote repair. Intriguingly certain neuron-intrinsic growth regulators are emerging as promising targets to both stimulate growth and relieve extrinsic inhibition of regeneration. This crossroads between the intrinsic and extrinsic aspects of spinal cord injury is a promising target for effective therapies for this unmet need.

Extracellular functions of 14-3-3 adaptor proteins.

14-3-3s are a family of adaptor proteins with a wide range of roles in cell signaling. Although they are primarily localized within the cytosol, 14-3-3s are also known to be present in the extracellular environment. Externalization of 14-3-3 can occur as a result of cell death or physiologically via release in exosomes. Interesting biological activities with relevance for tissue homeostasis and disease are now being described for extracellular 14-3-3s. Moreover, aminopeptidase N (APN) has been identified as a cell surface receptor for 14-3-3s. Here we review the array of bioactivities that have been ascribed to extracellular 14-3-3s and discuss applications as biomarkers and as targets for drug development.

14-3-3 adaptor protein-protein interactions as therapeutic targets for CNS diseases

&NA; 14‐3‐3s are a family of ubiquitously expressed adaptor proteins that regulate hundreds of functionally diverse ‘client proteins.’ In humans, there are seven isoforms with conserved structure and function. 14‐3‐3s typically bind to client proteins at phosphorylated serine/threonine motifs via a linear binding groove. Binding can have a variety of effects on the stability, activity and/or localization of the client protein. 14‐3‐3s are generating significant interest as potential drug targets for their involvement in cellular homeostasis and disease. They are especially abundant in the central nervous system (CNS) and are implicated in numerous CNS diseases, often through specific interactions with disease‐relevant client proteins. Several tool compounds that can modulate 14‐3‐3 interactions with client proteins to elicit therapeutic effects have recently been described. Here we offer a perspective on the functions of 14‐3‐3s in neurons and the potential development of drugs to therapeutically target 14‐3‐3 PPIs for CNS diseases. Graphical abstract Figure. No caption available.

Maximizing functional axon repair in the injured central nervous system: Lessons from neuronal development

The failure of damaged axons to regrow underlies disability in central nervous system injury and disease. Therapies that stimulate axon repair will be critical to restore function. Extensive axon regeneration can be induced by manipulation of oncogenes and tumor suppressors; however, it has been difficult to translate this into functional recovery in models of spinal cord injury. The current challenge is to maximize the functional integration of regenerating axons to recover motor and sensory behaviors. Insights into axonal growth and wiring during nervous system development are helping guide new approaches to boost regeneration and functional connectivity after injury in the mature nervous system. Here we discuss our current understanding of axonal behavior after injury and prospects for the development of drugs to optimize axon regeneration and functional recovery after CNS injury. Developmental Dynamics 247:18–23, 2018.

Targeting 14-3-3 adaptor protein-protein interactions to stimulate central nervous system repair

The goal of developing treatments for central nervous system (CNS) injuries is becoming more attainable with the recent identification of various drugs that can repair damaged axons. These discoveries have stemmed from screening efforts, large expression datasets and an improved understanding of the cellular and molecular biology underlying axon growth. It will be important to continue searching for new compounds that can induce axon repair. Here we describe how a family of adaptor proteins called 14-3-3s can be targeted using small molecule drugs to enhance axon outgrowth and regeneration. 14-3-3s bind to many functionally diverse client proteins to regulate their functions. We highlight the recent discovery of the axon-growth promoting activity of fusicoccin-A, a fungus-derived small molecule that stabilizes 14-3-3 interactions with their client proteins. Here we discuss how fusicoccin-A could serve as a starting point for the development of drugs to induce CNS repair.

Maximizing functional axon repair in the injured CNS – lessons from neuronal development

The failure of damaged axons to regrow underlies disability in central nervous system injury and disease. Therapies that stimulate axon repair will be critical to restore function. Extensive axon regeneration can be induced by manipulation of oncogenes and tumor suppressors; however, it has been difficult to translate this into functional recovery in models of spinal cord injury. The current challenge is to maximize the functional integration of regenerating axons to recover motor and sensory behaviors. Insights into axonal growth and wiring during nervous system development are helping guide new approaches to boost regeneration and functional connectivity after injury in the mature nervous system. Here we discuss our current understanding of axonal behavior after injury and prospects for the development of drugs to optimize axon regeneration and functional recovery after CNS injury. Developmental Dynamics 247:18–23, 2018.

Properties of human central nervous system neurons in a glia-depleted (isolated) culture system.

BACKGROUND Current methods for studying human neurons depend on a feeder layer of astroglia supplemented with animal serum to support the growing neurons. These requirements undermine many of the advantages provided by in vitro cell culture approaches when compared with more complex in vivo techniques. NEW METHOD Here, we identified a reliable marker (MHCI) that allows for direct isolation of primary neurons from fetal human brain. We utilized a magnetic labeling and isolation technique to separate neurons from other neural cells. We established a defined condition, omitting the astroglial supports that could maintain the human neurons for varying amounts of time. RESULTS We showed that the new method significantly improved the purity of human neurons in culture without the need for further chemical/mechanical enrichment. We demonstrated the suitability of these neurons for functional studies including Rho-kinase dependent regulation of neurite outgrowth and ensheathment in co-cultures with oligodendrocyte progenitor cells derived from fetal human brain. COMPARISON WITH EXISTING METHODS The accountability for neuron-only seeding and the controllable density allows for better neuronal maturation and better visualization of the different neuronal compartments. The higher purity culture constitutes an effective system to study and screen for compounds that impact neuron biology without potential confounding effects from glial crowding. CONCLUSIONS High purity human neurons generated using the improved method will enable enhanced reliability in the discovery and development of drugs with neuroregenerative and neuroprotective activity.

Axotomy-Induced Ganglioside Processing: A Mediator of Axon Regeneration Restricted to the PNS

It has long been observed that axons in the CNS have a poor ability to regenerate, while peripheral axons regenerate readily. Characterizing the molecular determinants that allow peripheral axons to regenerate might identify therapeutic strategies that may stimulate nerve regeneration and functional