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

km23-1 was previously identified as a TGF beta-receptor interacting protein that was phosphorylated on serines after TGF beta stimulation. In the current report, we examined the role of km23-1 phosphorylation in the downstream effects of TGF beta/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 TGF beta stimulation. Taken together, our results demonstrate that TGF beta 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 (573-km23-1) after TGF beta stimulation, immunoprecipitation (IP)/blot analyses were performed, which demonstrate that TGF beta regulates complex formation between the PKA regulatory subunit RIP and km23-1 in vivo. In addition, an S73A mutant of km23-1 (S73A-km23-1), which could not be phosphorylated by PKA, inhibited TGF beta 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 TGF beta, with 573-km23-1 being required for the CRE-dependent TGF beta stimulation of fibronectin (FN) transcription. Collectively, our results demonstrate for the first time that TGF beta/PKA phosphorylation of km23-1 on S73 is required for ARE- and CRE-mediated downstream events that include FN induction. (C) 2013 Published by Elsevier Inc.