title: 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 β-tubulin, P19 cells demonstrated a large, transient increase in both mRNA and protein for the endogenous protein kinase inhibitor (PKI)β. PKIβ-targeted shRNA constructs both reduced the levels of PKIβ 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 PKIβ protein, and this rescue required the PKA-specific inhibitory sequence of the PKIβ protein. PKIβ 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 PKIβ. Our results define a novel requirement for PKIβ and its inhibition of PKA during neuronal differentiation of P19 cells.
authors.affiliation: Neuroscience Graduate Program, University of Michigan, Ann Arbor, Michigan 48109-2200, USA.
authors.forename: Holly S
authors.initials: HS
authors.lastname: Huang
authors.forename: David L
authors.initials: DL
authors.lastname: Turner
authors.forename: Robert C
authors.initials: RC
authors.lastname: Thompson
authors.forename: Michael D
authors.initials: MD
authors.lastname: Uhler
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D050882:CREB-Binding Protein / Q000378:metabolism; D002277:Carcinoma / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics*; D002461:Cell Line, Transformed; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013873:Thionucleotides / Q000494:pharmacology; D014162:Transfection; D014404:Tubulin / Q000378:metabolism
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D050882:CREB-Binding Protein; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013873:Thionucleotides; D014404:Tubulin; C499317:Ascl1 protein, mouse; C462941:beta3 tubulin, mouse; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP