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

Abstract

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

DYNLRB1 is Essential for Dynein Mediated Transport and Neuronal Survival

“…We have characterized a new conditional knockout model for the DYNLRB1 subunit of dynein and use this model to show that DYNLRB1 is required for retrograde axonal transport of a broad spectrum of cargos in sensory axons, as well as for neuronal survival. These findings were surprising given the notion that DYNRLB1 should function mainly as a binding adaptor for TGFβ signaling complexes and other specific cargos [11,[14][15][16][17][18], whereas our data suggests that DYNLRB1 is essential for dynein-mediated axonal transport of lysosomes, late endosomes and signaling endosomes.…”

“…DYNLRB protein sequence is highly conserved among different species; with two isoforms, DYNLRB1 and DYNLRB2, sharing 98% sequence similarity in mammals [13]. Mammalian DYNLRB1 has been studied mainly as an adaptor linking specific modules to the dynein complex, including SMAD2 complexes activated by TGFβ receptors [11,[14][15][16][17][18] and Rab6 or N-acetyl-Dglucosamine kinase (NAGK) for interactions with the Golgi compartment [19][20][21].…”

KDEL receptor regulates secretion by lysosome relocation- and autophagy-dependent modulation of lipid-droplet turnover

“…2, 3), we analysed the presence of putative PKA phosphorylation sites in DynLRB1 that could regulate its function in this process. We found Serine-13 (S13) and S73 as good candidates, but S13 showed a low phosphorylation score (0.5) and was in fact discarded recently as a PKA target 32 . Instead, a large phospho-proteomic analysis identified S73 33 , which seems to be effectively phosphorylated by PKA 32 .…”

Decreased Tumor Progression and Invasion by a Novel Anti-Cell Motility Target for Human Colorectal Carcinoma Cells

“…Along these lines, we have shown that PKA can directly phosphorylate km23-1 on specific serine residues, which is required for TGFβ effects on downstream signaling of cAMP-responsive element (CRE)-dependent events (ie, FN) and Smad/activin-responsive element (ARE) activity [37]. Thus, km23-1 effects on Ezrin, as well as on other AKAP scaffolds, may involve PKA activity.…”

“…For example, km23-1 is required for TGFβ1 production through dynein-independent Ras/ERK/Jun N-terminal kinase (JNK) pathways in TGFβ-sensitive epithelial cells [35]. This multifunctional regulator also plays a critical dynein-dependent role in Smad2 signaling in early endosomes [34], and it is regulated by protein kinase A (PKA) activation [37]. Depletion of km23-1 not only reduces fibronectin (FN) expression, but also RhoA activity [30], further demonstrating the critical nature of km23-1's regulatory functions.…”