Caveolin-1 Phosphorylation Is Essential for Axonal Growth of Human Neurons Derived From iPSCs.

Proper axonal growth and guidance is essential for neuron differentiation and development. Abnormal neuronal development due to genetic or epigenetic influences can contribute to neurological and mental disorders such as Down syndrome, Rett syndrome, and autism. Identification of the molecular targets that promote proper neuronal growth and differentiation may restore structural and functional neuroplasticity, thus improving functional performance in neurodevelopmental disorders. Using differentiated human neuronal progenitor cells (NPCs) derived from induced pluripotent stem cells (iPSCs), the present study demonstrates that during early stage differentiation of human NPCs, neuron-targeted overexpression constitutively active Rac1 (Rac1CA) and constitutively active Cdc42 (Cdc42CA) enhance expression of P-Cav-1, T-Cav-1, and P-cofilin and increases axonal growth. Similarly, neuron-targeted over-expression of Cav-1 (termed SynCav1) increases axonal development by increasing both axon length and volume. Moreover, inhibition of Cav-1(Y14A) phosphorylation blunts Rac1/Cdc42-mediated both axonal growth and differentiation of human NPCs and SynCav1(Y14A)-treated NPCs exhibited blunted axonal growth. These results suggest that: (1) SynCav1-mediated dendritic and axonal growth in human NPCs is dependent upon P-Cav-1, (2) P-Cav-1 is necessary for proper axonal growth during early stages of neuronal differentiation, and (3) Rac1/Cdc42CA-mediated neuronal growth is in part dependent upon P-Cav-1. In conclusion, Cav-1 phosphorylation is essential for human neuronal axonal growth during early stages of neuronal differentiation

Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells

Engineering Pak1 Allosteric Switches

P21-activated kinases (PAKs) are important regulators of cell motility and morphology. It has been challenging to interrogate their functions because cells adapt to genetic manipulation of PAK, and because inhibitors act on multiple PAK isoforms. Here we describe genetically encoded PAK1 analogues that can be selectively activated by the membrane-permeable small molecule rapamycin. An engineered domain inserted away from the active site responds to rapamycin to allosterically control activity of the PAK1 isoform. To examine the mechanism of rapamycin-induced PAK1 activation, we used molecular dynamics with graph theory to predict amino acids involved in allosteric communication with the active site. This analysis revealed allosteric pathways that were exploited to generate kinase switches. Activation of PAK1 resulted in transient cell spreading in metastatic breast cancer cells, and long-term dendritic spine enlargement in mouse hippocampal CA1 neurons

Kinome-wide functional analysis highlights the role of cytoskeletal remodeling in somatic cell reprogramming

The creation of induced pluripotent stem cells (iPSCs) from somatic cells by ectopic expression of transcription factors has galvanized the fields of regenerative medicine and developmental biology. Here, we report a kinome-wide RNAi-based analysis to identify kinases that regulate somatic cell reprogramming to iPSCs. We prepared 3,686 small hairpin RNA (shRNA) lentiviruses targeting 734 kinase genes covering the entire mouse kinome and individually examined their effects on iPSC generation. We identified 59 kinases as barriers to iPSC generation and characterized seven of them further. We found that shRNA-mediated knockdown of the serine/threonine kinases TESK1 or LIMK2 promoted mesenchymal-to-epithelial transition, decreased COFILIN phosphorylation, and disrupted Actin filament structures during reprogramming of mouse embryonic fibroblasts. Similarly, knockdown of TESK1 in human fibroblasts also promoted reprogramming to iPSCs. Our study reveals the breadth of kinase networks regulating pluripotency and identifies a role for cytoskeletal remodeling in modulating the somatic cell reprogramming process