Regulation of Gli1 Localization by the cAMP/Protein Kinase A Signaling Axis through a Site Near the Nuclear Localization Signal

Abstract

The hedgehog (Hh) pathway plays a critical role during development of embryos and cancer. Although the molecular basis by which protein kinase A (PKA) regulates the stability of hedgehog downstream transcription factor cubitus interruptus, the Drosophila homologue of vertebrate Gli molecules, is well documented, the mechanism by which PKA inhibits the functions of Gli molecules in vertebrates remains elusive. Here, we report that activation of PKA retains Gli1 in the cytoplasm. Conversely, inhibition of PKA activity promotes nuclear accumulation of Gli1. Mutation analysis identifies Thr 374 as a major PKA site determining Gli1 protein localization. In the three-dimensional structure, Thr 374 resides adjacent to the basic residue cluster of the nuclear localization signal (NLS). Phosphorylation of this Thr residue is predicted to alter the local charge and consequently the NLS function. Indeed, mutation of this residue to Asp (Gli1/T374D) results in more cytoplasmic Gli1 whereas a mutation to Lys (Gli1/T374K) leads to more nuclear Gli1. Disruption of the NLS causes Gli1/T374K to be more cytoplasmic. We find that the change of Gli1 localization is correlated with the change of its transcriptional activity. These data provide evidence to support a model that PKA regulates Gli1 localization and its transcriptional activity, in part, through modulating the NLS function.

Activation of the hedgehog-signaling pathway in human cancer and the clinical implications

“…There are several ways to regulate the activity of Gli transcription factors. First, nuclear-cytoplasmic shuttling of Gli molecules is tightly regulated (Kogerman et al, 1999;Barnfield et al, 2005;Sheng et al, 2006;Stecca et al, 2007). For example, protein kinase A is shown to retain Gli1 proteins in the cytoplasm (through a PKA site in the nuclear localization signal peptide) whereas active Ras signaling promotes Gli nuclear localization (Stecca et al, 2007).…”

Suppressing Wnt Signaling by the Hedgehog Pathway through sFRP-1

“…For retrovirus infection, pLNCXGli1 (MYC-tagged) was transfected into Phoenix packaging cells using FuGENE 6 (Roche Applied Science). Supernatant was collected 24 -48 h post-transfection (24). Empty pLNCX vector was used to produce the control retrovirus.…”

“…Different dilutions were used for different primary antibodies: anti-sFRP-1, 1:1,000; anti-␤-catenin, 1:1,000; anti-MYC 9B11, 1:5,000; and anti-␤ actin, 1:5,000. The protein bands were visualized by enhanced chemiluminescence (24).…”

Inversion of Sonic hedgehog action on its canonical pathway by electrical activity

“…To determine the mechanisms by which Shh inhibits Gli activity in the spinal cord, we investigated different aspects of Gli activity regulation that are known to be mediated by PKA, including posttranslational processing and subcellular localization (12)(13)(14)(15)(16)(17). We evaluated the processing of Gli2/3 in the embryonic spinal cord by assessing the relative levels of full-length and cleaved (repressor) forms of endogenous Gli3 and exogenously expressed mGli2-GFP (35).…”

“…Despite persistence of Shh gradient and increased Shh levels as spinal cord development progresses (3,10), Gli activity is switched off (3,4) by unknown mechanisms. Some known inhibitory factors of Gli activity include Patched1, which operates in a negative feedback loop (11), and cAMP-dependent protein kinase (PKA), which acts by regulating Gli subcellular localization (12)(13)(14)(15), processing, and degradation (16,17).…”