Protein kinase D signaling through substrate phosphorylation

Protein kinase D (PKD) is a family of serine/threonine-specific protein kinases comprised of three isoforms: PKD1/PKCmicro, PKD2, and PKD3/PKCv. PKD contains two cysteine-rich domains that bind DAG, a PH domain that serves an autoinhibitory function, and a kinase domain. PKD localizes to the cytosol, nucleus, Golgi complex, and plasma membrane, where it regulates a variety of cellular processes ranging from cell proliferation, cell survival, Golgi organization, vesicle trafficking, and immune cell responses (Rykx et al., 2003; Van Lint et al., 2002). To date, only a small number of PKD substrates have been identified, some of which include the neuronal membrane protein Kidins220 (Iglesias et al., 2000), the Ras effector RIN1 (Wang et al., 2002), class II histone deacetylases HDACs 5 (Vega et al., 2004) and 7 (Dequiedt et al., 2005), the lipid kinase PI4KIIIbeta (Hausser et al., 2005), and the ceramide transport protein CERT (Fugmann et al., 2007). Given the various cellular responses attributed to this protein, we set out to identify additional PKD substrates.;A substrate-directed phospho-specific antibody that recognizes the consensus motif preferred by PKD (PKD pMOTIF) was used to identify novel PKD substrates (Doppler et al., 2005). The PKD consensus motif was defined through three independent studies and this information was considered when designing the peptide antigen, LXR(Q/K/E/M)(M/L/K/E/Q/A) S*XXXX, used to produce the PKD pMOTIF antibody (Doppler et al., 2005; Hutti et al., 2004; Nishikawa et al., 1997). By combining in vitro biochemical and in silico screening approaches with the substrate-directed PKD pMOTIF antibody, we were able to identify oxysterol-binding protein (OSBP) and signal-induced proliferation-associated 1-like protein 1 (SIPA1L1) as novel PKD substrates.;PKD phosphorylation of OSBP inhibits its 25-hydroxycholesterol-induced Golgi localization. These findings reveal PKD as the first identified upstream kinase to regulate OSBP localization. 25OH-mediated OSBP localization regulates sphingomyelin synthesis, demonstrating an additional mechanism in which PKD regulates Golgi lipid homeostasis. PKD phosphorylation of SIPA1L1 and binding through a PDZ domain-interaction regulates SIPA1L1 proteasomal degradation. The cellular abundance of SIPA1L1 protein is critical to its function in E6-induced mammary epithelial cell transformation and neuronal cell morphology, thus implicating PKD in regulation of these biological processes.