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

Authors

  • Holly S. Huang,

    1. Neuroscience Graduate Program, University of Michigan, Ann Arbor, Michigan, USA
    2. Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, Michigan, USA
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  • David L. Turner,

    1. Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, Michigan, USA
    2. Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA
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  • Robert C. Thompson,

    1. Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, Michigan, USA
    2. Department of Psychiatry, University of Michigan, Ann Arbor, Michigan, USA
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  • Michael D. Uhler

    1. Molecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, Michigan, USA
    2. Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan, USA
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Address correspondence and reprint requests to Michael D. Uhler, Molecular and Behavioral Neuroscience Institute, University of Michigan, 109 Zina Pitcher Pl, Ann Arbor, MI 48109-2200, USA. E-mail: muhler@umich.edu

Abstract

J. Neurochem (2012) 120, 667–683.

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.

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