Repair after injury to the adult mammalian central nervous system (CNS) is hindered by inhibitory proteins, including the myelin associated inhibitors (MAIs): NogoA, MAG, and OMgp. Blocking the function of MAIs and their receptors enhances axon regrowth following injury. These findings suggest that therapeutic control of these inhibitors may be a strategy for regulating axon outgrowth and plasticity, leading to restoration of neuronal connections lost in response to injury or disease. Thus, functional recovery after CNS injury is limited by MAIs, and small molecule compounds that can circumvent MAI inhibition are likely to enhance functional recovery after stroke or spinal cord injury. The precise intracellular molecular signaling mechanisms of MAIs are not well understood. Toward this goal, it is demonstrated here that the multi-domain scaffold protein POSH assembles a distinct signaling module composed of the mixed-lineage kinase LZK, the actin-myosin regulatory protein Shroom3, and Rho-associated kinase, ROCK. Through the receptor PirB, the POSH complex mediates growth inhibitory signals from extracellular NogoA and MAG, as well as cell autonomous NogoA signaling. PirB associates with the protein tyrosine phosphatase, Shp2, and we show that phosphatase activity of Shp2 is required for axonal growth inhibition. In addition, NogoA stimulation promotes trapping of LZK with Shp2, suggesting LZK is a potential substrate for dephosphorylation by Shp2. Lastly, interference with the function of any member of the POSH complex results in enhanced growth on MAIs, suggesting that chemicals that target protein-protein interactions within the POSH complex will reduce the inhibitory action of MAIs, facilitating axon outgrowth in the CNS. Towards this goal, high-throughput screening in the Center for Chemical Genomics at the University of Michigan has identified potential inhibitors of the Shroom3-ROCK interaction. Collectively, these studies delineate an intracellular signaling pathway emanating from MAIs through the receptor PirB to the POSH complex. Further insight into the molecular signaling mechanisms of this pathway may provide novel therapeutic targets for axonal repair following CNS injury.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/93965/1/dicksonh_1.pdf