PKCδ regulates the stimulation of vascular endothelial factor mRNA translation by angiotensin II through hnRNP K
Introduction
Angiotensin II is a mediator of renal injury in diabetic nephropathy and other chronic kidney diseases [1]. Treatment of proximal tubular epithelial cells (MCT cells) with 1 nM of angiotensin II (Ang II) stimulates synthesis of vascular endothelial growth factor (VEGF) through increased translation of its mRNA [2]. This was shown to be due to stimulation of cap-dependent translation initiation, which takes place at the 5′ untranslated region (UTR) of the mRNA [2]. Additional studies showed that VEGF mRNA translation was positively regulated by binding of heterogeneous ribonucleoprotein K (hnRNP K) to the 3′ UTR of the VEGF mRNA [3]. Activation of c-src by Ang II is critical to the binding of hnRNP K to VEGF mRNA, through phosphorylation of the latter on Ser302, which is a target of PKCδ [3].
There are six known tyrosine residues in hnRNP K that undergo phosphorylation by members of the c-src family [4]. Phosphorylation of hnRNP K on tyrosine residues by c-src is believed to generate several c-src homology domain 2 (SH2)-docking sites, which allow the protein to interact with various SH2-containing proteins, such as Lck and PKCδ. Although the tyrosine residues affected in hnRNP K lie outside the KH domains responsible for RNA binding, tyrosine phosphorylation of hnRNP K is generally considered to inhibit its binding to specific RNAs. For instance, Ostareck-Lederer et al. have shown that hnRNP K and the c-src kinase specifically interact with each other, leading to c-src-mediated tyrosine phosphorylation of hnRNP K in vivo and in vitro [4]. c-src-mediated phosphorylation reversibly inhibits the binding of hnRNP K to the differentiation control element (DICE) of the LOX mRNA 3′ untranslated region in vitro and specifically de-represses the translation of DICE-bearing mRNAs in vivo [4]. Similarly, Ostrowski et al. have shown that hnRNP K protein is constitutively bound to many mRNAs in vivo, and that many hnRNP K-mRNA complexes are disrupted by tyrosine phosphorylation [5]. Interestingly, states of enhanced cell proliferation were associated with increased levels of hnRNP K tyrosine phosphorylation [6].
There are four serine residues in hnRNP K that undergo phosphorylation by extracellular signal-regulated kinase (ERK), c-Jun N terminal kinase (JNK), and members of the protein kinase C (PKC) family. It has been shown that serine phosphorylation of hnRNP K is RNA- and DNA-dependent, i.e., RNA- or DNA-bound hnRNP K is a preferred substrate for protein kinases [7]. Serine phosphorylation of hnRNP K is believed to alter its intracellular distribution, leading to cytoplasmic accumulation [8], [9]. Phosphorylation of hnRNP K by ERK also leads to inhibition of mRNA translation [8].
hnRNP K has been shown to act as a docking platform that allows Lck, a member of the c-src family, to interact with a member of another kinase cascade, PKCδ to control the activity of a translation eukaryotic elongation factor, eEF1A (EF-1α) [4], [5], [7], [10]. While bound to RNA, hnRNP K is prevented from direct interaction with PKCδ [10]. The binding of Lck to hnRNP K enhances its activity resulting in phosphorylation of hnRNP K on additional tyrosine residues, causing dissociation of hnRNP K protein from RNA [4], [5] and allowing the recruitment of PKCδ [10]. Following binding, the DAG-primed PKCδ is further induced through tyrosine phosphorylation by the activated Lck docked next to it [10]. Activated PKCδ not only targets Ser302 on hnRNP K [10] but also phosphorylates effectors either bound to K protein or present in its microenvironment through imposed proximity. EF-1α binds hnRNP K [11] and is a substrate of PKCδ [12]. Therefore, PKCδ-mediated phosphorylation of EF-1α could occur in the context of hnRNP K, and contribute to the activation of the translational machine.
Although the importance of hnRNP K phosphorylation by PKCδ has been established [3], [10], the functional consequences of phosphorylation of hnRNP K by PKCδ are not known. Also unknown is whether similar association between hnRNP K and PKCdelta occurs in vivo in the kidney in a disease state. In this study, we sought to investigate the role of PKCδ-mediated phosphorylation of hnRNP K in the regulation of VEGF mRNA translation by Ang II in MCT cells and assess the association between the two proteins in vivo in the kidney tissue of mice with type 2 diabetes.
Section snippets
Cell culture
SV40-immortalized murine proximal tubular epithelial cells (MCT) were provided by Dr. Eric Neilson, Vanderbilt University, Nashville, TN. MCT cells in culture express in vivo characteristics of proximal tubular epithelial cells. [13]. The cells were grown in Dulbecco's minimal essential medium (DMEM) containing 5 mM glucose and 10% FBS [2], [3]. Confluent monolayers of cells were serum-deprived in DMEM for 18 h before treatment.
Animal experiments
C57BLKsJ lepr−/− db/db mice and their lean littermate controls
Results
Ang II stimulates PKCδ activity in MCT cells with a time-course corresponding to that of increased VEGF synthesis [3]. Phosphorylation of hnRNP K on Ser302, a known target of PKCδ [10], positively correlated with its binding to VEGF mRNA, and knockdown of hnRNP K expression by RNA interference significantly reduced Ang II stimulation of VEGF synthesis [3]. However, the requirement for PKCδ for either hnRNP K phosphorylation on Ser302 or for Ang II-induction of VEGF synthesis has not been
Discussion
Our data demonstrate the following: (1) Inhibition of PKCδ expression reduces Ang II stimulation of VEGF synthesis, similar to inhibition of hnRNP K expression. (2) hnRNP K recruits both c-src and PKCδ activated by Ang II that allows phosphorylation and activation of PKCδ by c-src. (3) PKCδ, in turn, phosphorylates and activates hnRNP K, allowing its binding to VEGF mRNA (4) Following Ang II stimulation, both hnRNP K binding to VEGF mRNA and its translation are PKCδ dependent. (5) In kidney
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