How scaffold proteins integrate signaling pathways with cytoskeletal components to drive axon outgrowth is not well understood. We report here that the multidomain scaffold protein Plenty of SH3s (POSH) regulates axon outgrowth. Reduction of POSH function by RNA interference (RNAi) enhances axon outgrowth in differentiating mouse primary cortical neurons and in neurons derived from mouse P19 cells, suggesting POSH negatively regulates axon outgrowth. Complementation analysis reveals a requirement for the third Src homology (SH) 3 domain of POSH, and we find that the actomyosin regulatory protein Shroom3 interacts with this domain of POSH. Inhibition of Shroom3 expression by RNAi leads to increased process lengths, as observed for POSH RNAi, suggesting that POSH and Shroom function together to inhibit process outgrowth. Complementation analysis and interference of protein function by dominant-negative approaches suggest that Shroom3 recruits Rho kinase to inhibit process outgrowth. Furthermore, inhibition of myosin II function reverses the POSH or Shroom3 RNAi phenotype, indicating a role for myosin II regulation as a target of the POSH-Shroom complex. Collectively, these results suggest that the molecular scaffold protein POSH assembles an inhibitory complex that links to the actin-myosin network to regulate neuronal process outgrowth.

Myelin-derived inhibitors limit axon outgrowth and plasticity during development and in the adult mammalian CNS. Nogo66, a functional domain of the myelin-derived inhibitor NogoA, signals through the PirB receptor to inhibit axon outgrowth. The signaling pathway mobilized by Nogo66 engagement of PirB is not well understood. We identify a critical role for the scaffold protein Plenty of SH3s (POSH) in relaying process outgrowth inhibition downstream of Nogo66 and PirB. Blocking the function of POSH, or two POSH-associated proteins, leucine zipper kinase (LZK) and Shroom3, with RNAi in cortical neurons leads to release from myelin and Nogo66 inhibition. We also observed autocrine inhibition of process outgrowth by NogoA, and suppression analysis with the POSH-associated kinase LZK demonstrated that LZK operates downstream of NogoA and PirB in a POSH-dependent manner. In addition, cerebellar granule neurons with an RNAi-mediated knockdown in POSH function were refractory to the inhibitory action of Nogo66, indicating that a POSHdependent mechanism operates to inhibit axon outgrowth in different types of CNS neurons. These studies delineate an intracellular signaling pathway for process outgrowth inhibition by Nogo66, comprised of NogoA, PirB, POSH, LZK, and Shroom3, and implicate the POSH complex as a potential therapeutic target to enhance axon outgrowth and plasticity in the injured CNS.

G protein-coupled receptor (GPCR) kinases (GRKs) were discovered by virtue of their ability to phosphorylate activated GPCRs. They constitute a branch of the AGC kinase superfamily, but their mechanism of activation is largely unknown. To initiate a study of GRK2 activation, we sought to identify sites on GRK2 remote from the active site that are involved in interactions with their substrate receptors. Using the atomic structure of GRK2 in complex with Gβγ as a guide, we predicted that residues on the surface of the kinase domain that face the cell membrane would interact with the intracellular loops and carboxyl (C) terminal tail of the GPCR. Our study focused on two regions: the kinase large lobe and an extension of the kinase domain known as the C-tail. Conserved residues in the GRK2 large lobe whose side-chains are solvent exposed and facing the membrane were targeted for mutagenesis. Residues in the C-tail of GRK2, although not ordered in the crystal structure, were also targeted because this region has been implicated in receptor binding and in the regulation of AGC kinase activity. Four substitutions out of twenty, all within or adjacent to the C-tail, resulted in significant deficiencies in the ability of the enzyme to phosphorylate activated GPCRs, rhodopsin and the β 2 -adrenergic receptor (β 2 AR). The mutant exhibiting the most dramatic impairment, V477D, also showed significant defects in phosphorylation of non-receptor substrates. Interestingly, Michaelis-Menten kinetics suggested that V477D had 12-fold lower k cat , but no changes in K M , suggesting a defect in acquisition or stabilization of the closed state of the kinase domain. V477D was also resistant to activation by agonist-treated β 2 AR. Therefore, Val477 and other residues in the C-tail are expected to play a role in the activation of GRK2 by GPCRs.G protein-coupled receptors (GPCRs), distinguished by their seven transmembrane helices and their ability to couple to heterotrimeric G proteins (Gαβγ), are activated by hormones, neurotransmitters and sensory signals, and regulate many diverse processes. Receptor stimulation catalyzes the binding of GTP to the Gα subunit, allowing Gα and Gβγ to directly † This work was funded by the National Science Foundation grants MCB0315888 and MCB0744739 (to RSM), National Institutes of Health grants HL086865 and HL071818 and American Heart Association Scientist Development Grant 0235273N (to JJGT), and a summer undergraduate research fellowship from the Northeast Affiliate of the American Heart Association (to EDB). *To whom correspondence should be addressed. Biology Department, Siena College, Morrell Science Center, 515 Loudon Road, Loudonville, NY 12211, Tel: (518) 783-2462; Fax: (518) ; email: sternemarr@siena.edu. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 May 26. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript modulate effectors such as adenylyl cyclase, phospholipase Cβ, cGMP phosphodiesterase, ion channels and ...

BackgroundInhibitory molecules in the adult central nervous system, including NogoA, impede neural repair by blocking axon outgrowth. The actin-myosin regulatory protein Shroom3 directly interacts with Rho kinase and conveys axon outgrowth inhibitory signals from Nogo66, a C-terminal inhibitory domain of NogoA. The purpose of this study was to identify small molecules that block the Shroom3–Rho kinase protein–protein interaction as a means to modulate NogoA signaling and, in the longer term, enhance axon outgrowth during neural repair.ResultsA high throughput screen for inhibitors of the Shroom3–Rho kinase protein–protein interaction identified CCG-17444 (Chem ID: 2816053). CCG-17444 inhibits the Shroom3–Rho kinase interaction in vitro with micromolar potency. This compound acts through an irreversible, covalent mechanism of action, targeting Shroom3 Cys1816 to inhibit the Shroom3–Rho kinase protein–protein interaction. Inhibition of the Shroom3–Rho kinase protein–protein interaction with CCG-17444 counteracts the inhibitory action of Nogo66 and enhances neurite outgrowth.ConclusionsThis study identifies a small molecule inhibitor of the Shroom3–Rho kinase protein–protein interaction that circumvents the inhibitory action of Nogo66 in neurons. Identification of a small molecule compound that blocks the Shroom3–Rho kinase protein–protein interaction provides a first step towards a potential new strategy for enhancing neural repair.