Protein kinase D activity controls endothelial nitric oxide synthesis

Abstract

We have recently been made aware of concerns regarding some of the data in Fig. 4. After discussion with the corresponding author, Ignacio Rodríguez-Crespo, we have referred this matter to Dr Rodríguez-Crespo's institute. Journal of Cell Science is publishing this note to make readers aware of the issue, and we will provide further information once it has been resolved.This course of action follows the advice set out by COPE (Committee on Publication Ethics), of which Journal of Cell Science is a member. Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone.

Emergency Spatiotemporal Shift: The Response of Protein Kinase D to Stress Signals in the Cardiovascular System

“…Protein Kinase D1 has also been implicated in the phosphorylation and regulation of constitutive nitric oxide synthases ( e ndothelial and n euronal NOS; Aicart-Ramos et al, 2014; Sanchez-Ruiloba et al, 2014). In endothelial cells, VEGF activation of PKD promotes vasodilation and angiogenesis via S1179 phosphorylation, and activation, of eNOS.…”

Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase

“…Yeast Two-hybrid Assays-Essentially, we followed our own published protocol (30,31). We used plasmids containing GAL4 binding domain that were confronted with plasmids containing the GAL4 activation domain as described previously (29).…”

Binding of PDZ domains to the carboxy terminus of inducible nitric oxide synthase boosts electron transfer and NO synthesis

“…(B) COS‐7 cells were transfected with full‐length wild‐type iNOS alone (−) or together with full‐length CAP70 or EBP50. One day post‐transfection the medium was replaced and 48 h after transfection cells were harvested, sonicated for 5 s and conversion of [ 14 C]‐ l ‐Arg to [ 14 C]‐ l ‐Cit was analyzed [15]. The resulting [ 14 C]‐ l ‐Cit measurement obtained for non‐transfected cells was subtracted from each condition and the [ 14 C]‐ l ‐Arg to [ 14 C]‐ l ‐Cit conversion (%) was referred to cells transfected with iNOS to which a 100% conversion was assigned.…”

“…Every sample was analyzed in triplicate. The enzymatic activity of iNOS was determined using the Griess assay and also the conversion of [ 14 C]‐ l ‐arginine to [ 14 C‐]‐ l ‐citrulline conversion assay as previously reported [15,28]. The K m for l ‐Arg for wild‐type full‐length iNOS after the binding of CAP70 (12.6 μM) or EBP50 (25 μM) to the C‐terminal end of the enzyme was calculated using the Griess assay as previously described [28,29].…”

“…Every sample was analyzed in triplicate. The enzymatic activity of iNOS was determined using the Griess assay and also the conversion of [ 14 C]-L-arginine to [ 14 C-]-L-citrulline conversion assay as previously reported [15,28].…”