Requirement for protein kinase A in the phosphorylation of the TGFΒ receptor-interacting protein km23-1 as a component of TGFΒ downstream effects

Qunyan Jin, Yan Zhong, Kathleen M. Mulder

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Abstract

km23-1 was previously identified as a TGFΒ-receptor interacting protein that was phosphorylated on serines after TGFΒ stimulation. In the current report, we examined the role of km23-1 phosphorylation in the downstream effects of TGFΒ/protein kinase A (PKA) signaling. Using phosphorylation site prediction software, we found that km23-1 has two potential PKA consensus phosphorylation sites. In vitro kinase assays further demonstrated that PKA directly phosphorylates km23-1 on serine 73 (S73). Moreover, our results show that the PKA-specific inhibitor H89 diminishes phosphorylation of km23-1 on S73 after TGFΒ stimulation. Taken together, our results demonstrate that TGFΒ induction of PKA activity results in phosphorylation of km23-1 on S73. In order to assess the mechanisms underlying PKA phosphorylation of km23-1 on S73 (S73-km23-1) after TGFΒ stimulation, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGFΒ regulates complex formation between the PKA regulatory subunit RIΒ and km23-1 in vivo. In addition, an S73A mutant of km23-1 (S73A-km23-1), which could not be phosphorylated by PKA, inhibited TGFΒ induction of the km23-1-dynein complex and transcriptional activation of the activin-responsive element (ARE). Furthermore, our results show that km23-1 is required for cAMP-responsive element (CRE) transcriptional activation by TGFΒ, with S73-km23-1 being required for the CRE-dependent TGFΒ stimulation of fibronectin (FN) transcription. Collectively, our results demonstrate for the first time that TGFΒ/PKA phosphorylation of km23-1 on S73 is required for ARE- and CRE-mediated downstream events that include FN induction.

Original languageEnglish (US)
Pages (from-to)897-907
Number of pages11
JournalExperimental Cell Research
Volume319
Issue number6
DOIs
StatePublished - Jan 1 2013

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All Science Journal Classification (ASJC) codes

  • Cell Biology

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