, 2003), epidermal hair tip and sensory bristle formation in Drosophila ( Cong et al., 2001 and Geng Selleck Apoptosis Compound Library et al., 2000), and dendritic morphogenesis and tiling in the worm and fly ( Emoto et al., 2004, Emoto et al., 2006, Gallegos and Bargmann, 2004 and Han et al., 2012). The

two Trc homologs mammalian (Stk38) and NDR2 (Stk38l; referred to as NDR1/2) are ∼86% identical. Their biochemical activation has been well characterized with no difference between NDR1 and NDR2 reported ( Hergovich et al., 2006). NDR1 and NDR2 are broadly expressed in the mouse brain ( Devroe et al., 2004, Stegert et al., 2004 and Stork et al., 2004). NDR1 knockout mice have increased susceptibility to tumor formation, implicating NDR1 as tumor suppressor ( Cornils et al., 2010). NDR2

levels are increased in NDR1 knockout mice and may compensate for the absence of NDR1 ( Cornils et al., 2010). The potential roles of NDR1/2 in regulating mammalian neuronal morphogenesis are unknown. Despite the importance of the NDR1/2 kinase pathway in regulating cellular morphogenesis in eukaryotes, the downstream phosphorylation targets of NDR1/2 remain largely unknown, except for two substrates for the PLX4032 NDR1/2 yeast homolog Cbk1: Sec2p (Kurischko et al., 2008) and Ssd1p, a nonconserved protein (Jansen et al., 2009), and a recently identified NDR1/2 substrate p21 (Cornils et al., 2011). To elucidate the mechanism of NDR1/2 kinase actions in neurons, it is important to identify the direct phosphorylation targets of NDR1/2 and their functions in the brain. In this study, we investigated NDR1/2 function in cultured rat hippocampal neurons and in mouse cortical neurons in vivo

by perturbing its function via the expression of dominant negative and constitutively active NDR1/2 and siRNA. We found that NDR1/2 kinases limit dendrite branching and length in cultures and in vivo, analogous to the roles of their fly homolog Trc. Additionally, NDR1/2 kinases were also required aminophylline for mushroom spine synapse formation as NDR1/2 loss of function led to more immature spines, both in cultures and in vivo, as well as a reduction in the frequency of miniature excitatory postsynaptic currents (mEPSCs) in neuronal cultures. To uncover the direct targets of NDR1/2, which control dendrite branching and mushroom spine formation, we used chemical genetics to create a mutant NDR1 capable of uniquely utilizing an ATP analog not recognized by endogeneous protein kinases (Blethrow et al., 2008 and Shah et al., 1997). An advantage of this method is that it identifies not only the substrates but also the phosphorylation sites. We identified five potential NDR1 substrates in the mouse brain and chose two for functional validation. We show that one NDR1 substrate is another kinase, AP-2 associated kinase-1 (AAK1), which regulates dendritic branching as a result of NDR1 phosphorylation.