Ascl1-induced neuronal differentiation of P19 cells requires expression of a specific inhibitor protein of cyclic AMP-dependent protein kinase.

ABSTRACT: cAMP-dependent protein kinase (PKA) plays a critical role in nervous system development by modulating sonic hedgehog and bone morphogenetic protein signaling. In the current studies, P19 embryonic carcinoma cells were neuronally differentiated by expression of the proneural basic helix-loop-helix transcription factor Ascl1. After expression of Ascl1, but prior to expression of neuronal markers such as microtubule associated protein 2 and neuronal ?-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)?. PKI?-targeted shRNA constructs both reduced the levels of PKI? expression and blocked the neuronal differentiation of P19 cells. This inhibition of differentiation was rescued by transfection of a shRNA-resistant expression vector for the PKI? protein, and this rescue required the PKA-specific inhibitory sequence of the PKI? protein. PKI? played a very specific role in the Ascl1-mediated differentiation process as other PKI isoforms were unable to rescue the deficit conferred by shRNA-mediated knockdown of PKI?. Our results define a novel requirement for PKI? and its inhibition of PKA during neuronal differentiation of P19 cells.

SUBMITTER: Huang HS 

PROVIDER: S-EPMC3363362 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature