These studies uncover two downstream signaling pathways defined by a kinase (AAK1) and a GEF (Rabin8), which regulate complex neuronal dendritic and synaptic phenotypes orchestrated by NDR1/2. NDR1 and NDR2 transcripts

have been found in the brain by reverse transcription polymerase chain reaction (RT-PCR) and northern blot (Devroe et al., 2004 and Stegert et al., 2004), and NDR2 mRNA has been localized via in situ buy PD-0332991 hybridization in various brain regions, including the hippocampus and cortex (Stork et al., 2004). To determine the developmental profile of NDR1 and NDR2 expression, we probed brain lysates from postnatal day (P)5, P10, P15 and P20 via a mouse monoclonal antibody raised against NDR1 and a polyclonal antibody we generated that is specific for NDR2 (see Experimental Procedures). Both antibodies recognized a major protein band, which was present throughout development, at ∼55 KD (Figures 1A; Figure S1A available online). The NDR1 antibody did not recognize overexpressed NDR2, and the NDR2 antibody did not recognize overexpressed NDR1 in COS-7 cells, demonstrating their specificity (Figure S1B). Immunocytochemistry using these antibodies revealed that NDR1 and NDR2 are present in the cytoplasm

in hippocampal pyramidal neurons and in the cortex (Figure 1B and data not shown) and are found throughout the cell body and dendrites in dissociated hippocampal neurons in culture (Figure 1C). NDR1 was also present in the nucleus in agreement with previous reports (Millward et al., 1999; data not shown). In order to investigate NDR1/2′s cell autonomous Adriamycin in vitro function in dendrite development, we used three approaches. Dominant-negative or constitutively active NDR1/2 expression, siRNA knockdown of NDR1 and NDR2, and a chemical genetics approach to block NDR1 activity were used. NDR1 mutations used in this study are shown in Figures 1D and 1E. We found similar results with all three approaches. The biochemical activation mechanism of NDR kinases has been established. MST3 kinase phosphorylates NDR1/2 at its C-terminal hydrophobic

residue T444 to activate Phosphatidylinositol diacylglycerol-lyase it (Stegert et al., 2005). NDR1/2 can be activated by okadaic acid (OA) via inhibition of protein phosphatase 2A, facilitating phosphorylation at T444 and the autophosphorylation at S281 (Stegert et al., 2005). MOB1/2 binding to the N-terminal region of NDR kinases is required for the release of auto-inhibition and maximal activity (Bichsel et al., 2004). Autophosphorylation site S281 is critical for NDR1/2 kinase activity. In order to test NDR1/2′s role in dendrite development, we first generated dominant negative and constitutively active NDR1 mutants (Figures 1D and 1E). For dominant negative NDR1, we mutated Ser281 and Thr444 to Alanine (S281A; T444A, NDR1-AA) or catalytic lysine to alanine (K118A, NDR1-KD); both mutants have no kinase activity (Millward et al., 1999 and Stegert et al., 2004).