It is important to note that secretory membrane trafficking has been found to be critical for dendrite morphogenesis (Horton et al., 2005 and Ye et al., 2007). The additional potential substrates of NDR1/2 identified in our study could also affect vesicle trafficking. For instance, PI4KB can catalyze formation of phosphatidyl inositol 4 phosphate (PI4P), which is an intermediate in the formation of phosphorylated lipids, such as PI3,4 bisphosphate, PI4,5 bisphosphate, and PI3,4,5 trisphosphate (De Matteis et al., 2005). These phospholipids are known to affect membrane trafficking in post-Golgi and recycling membrane compartments (De Matteis et al., 2005).

Another potential substrate, Rab11fip5, is a member of Rab11 family interacting proteins (Horgan and McCaffrey, 2009), which could Epigenetic Reader Domain inhibitor affect membrane trafficking from recycling endosomes in dendrites (Wang et al., 2008). The chemical genetics and covalent capture method for kinase substrate identification is a powerful method for mapping of kinase signaling pathways with the unique advantage of phosphorylation site identification (Hertz et al., 2010 and Blethrow et al., 2008). This method also allows the identification of substrates from complex tissue homogenates, where the protein complexes may be better preserved in their natural state when compared to other methods that involve JQ1 cell line gel electrophoresis or protein

arrays. We were able to identify five mammalian candidate substrates and validated two of these functionally. Our screen identified the mammalian homolog of one of the yeast substrates Sec2p, confirming its effectiveness and establishing an evolutionarily conserved branch of NDR kinase signaling. Our technique offers an unbiased method for identifying kinase substrates from different tissues, developmental stages and pathological conditions. This approach would make it possible to determine how

NDR1/2 activity and targets are altered in pathologies, such as neurodevelopmental and neurodegenerative diseases and cancer. The use and care of rats and mice used in this study follows the guidelines of the UCSF Institutional Animal Care and Use Committee. Detailed Experimental Procedures can be found in the Supplemental Experimental Procedures. Hippocampal neurons were Astemizole cultured from E19 Long-Evans rats at 150,000/coverslip and maintained at serum-free B27-containing media. Plasmid transfections were done using Lipofectamine2000 (Invitrogen, Grand Island, NY, USA). Prk5 mammalian expression vector was used for mammalian expression of constructs in cultured neurons and in HEK293 cells. Small hairpins were cloned in a modified pGIPZ (Open Biosystems, Lafayette, CO, USA) for hippocampal cultures and pSuper vector for in utero electroporations. mEPSCs were recorded using whole-cell patch-clamping in the presence of 1 μM tetrodotoxin and 50 μM picrotoxin to isolate excitatory minis. E14.5–E15.