Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR

Schulz, Sebastian; Doller, Anke; Pendini, Nicole R.; Wilce, Jacqueline A.; Pfeilschifter, Josef; Eberhardt, W.

doi:10.1016/j.cellsig.2013.08.003

Cited by 25 publications

(26 citation statements)

References 55 publications

(88 reference statements)

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“…46 When the protein is phosphorylated, the negative charge of the protein is further increased (in absolute terms) and the repulsion effect would be even larger. In contrast to our findings, Schulz and coworkers 71 have recently reported a higher binding affinity of HuR S318D with U-rich bearing mRNA stretches. Plausible explanations arise from differences in length and degree of structure of U-rich long mRNAs and 5 0 -UUUUU-3 0 short RNA molecules and/or differences in number of RRMs making up HuR FL and RRM3 S318D species.…”

Section: Discussioncontrasting

confidence: 56%

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

et al. 2014

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Section: Discussioncontrasting

confidence: 56%

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

et al. 2014

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“…To this end, Schulz et al showed that the effect of PKC phosphorylation on HuR function is dependent on the specific site of phosphorylation. [24] Specifically, they used phosphor-mimetic mutants to show that only PKC phosphorylation of Ser221 mediates HuR nucleo-cytoplasmic shuttling, while phosphorylation at either Ser158 or Ser318 mediates RNA binding specificity. [24] Future work will be needed to identify the role that specific HuR phosphorylation sites play in myocyte hypertrophy.…”

Section: Discussionmentioning

confidence: 99%

Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy

Slone

Anthony

et al. 2016

Cellular Signalling

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The RNA binding protein Human antigen R (HuR) interacts with specific AU-rich domains in target mRNAs and is highly expressed in many cell types, including cardiomyocytes. However, the role of HuR in cardiac physiology is largely unknown. Our results show that HuR undergoes cytoplasmic translocation, indicative of its activation, in hypertrophic cardiac myocytes. Specifically, HuR cytoplasmic translocation is significantly increased in NRVMs (neonatal rat ventricular myocytes) following treatment with phenylephrine or angiotensin II, agonists of two independent Gαq-coupled GPCRs known to induce hypertrophy. This Gq-mediated HuR activation is dependent on p38 MAP kinase, but not canonical Gq-PKC signaling. Furthermore, we show that HuR activation is necessary for Gq-mediated hypertrophic growth of NRVMs as siRNA-mediated knockdown of HuR inhibits hypertrophy as measured by cell size and expression of ANF (atrial natriuretic factor). Additionally, HuR overexpression is sufficient to induce hypertrophic cell growth. To decipher the downstream mechanisms by which HuR translocation promotes cardiomyocyte hypertrophy, we assessed the role of HuR in the transcriptional activity of NFAT (nuclear factor of activated T cells), the activation of which is a hallmark of cardiac hypertrophy. Using an NFAT-luciferase reporter assay, we show an acute inhibition of NFAT transcriptional activity following pharmacological inhibition of HuR. In conclusion, our results identify HuR as a novel mediator of cardiac hypertrophy downstream of the Gq-p38 MAPK pathway, and suggest modulation of NFAT activity as a potential mechanism.

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“…Given that this peptide is known to be phosphorylated by PKC (20), it is assumed that serine-containing peptide number 14 in the FRET sensor will be phosphorylated in the presence of ATP. Aspartic acid has been successfully used with other PKC substrates to mimic phosphorylation (21)(22)(23). Hence, a phosphomimetic version of peptide number 14 was also created by mutating the alanine in the peptide to aspartic acid.…”

Section: Resultsmentioning

confidence: 99%

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

Sommese¹,

Sivaramakrishnan

2016

Journal of Biological Chemistry

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The overlapping network of kinase-substrate interactions provides exquisite specificity in cell signaling pathways, but also presents challenges to our ability to understand the mechanistic basis of biological processes. Efforts to dissect kinase-substrate interactions have been particularly limited by their inherently transient nature. Here, we use a library of FRET sensors to monitor these transient complexes, specifically examining weak interactions between the catalytic domain of protein kinase C␣ and 14 substrate peptides. Combining results from this assay platform with those from standard kinase activity assays yields four novel insights into the kinase-substrate interaction. First, preferential binding of non-phosphorylated versus phosphorylated substrates leads to enhanced kinase-specific activity. Second, kinase-specific activity is inversely correlated with substrate binding affinity. Third, high affinity substrates can suppress phosphorylation of their low affinity counterparts. Finally, the substrate-competitive inhibitor bisindolylmaleimide I displaces low affinity substrates more potently leading to substrate selective inhibition of kinase activity. Overall, our approach complements existing structural and biophysical approaches to provide generalizable insights into the regulation of kinase activity.The canonical role of a protein kinase is to recognize, bind, and phosphorylate specific serine, threonine, or tyrosine residues on a substrate protein (1, 2). Given that kinases are one of the largest gene families in eukaryotes and are involved in nearly every cellular function (3), significant work has focused on understanding the mechanisms that dictate substrate-kinase pairings (4). Although the catalytic domains of eukaryotic kinases are structurally conserved (5, 6), the local environment around the substrate binding pocket of the kinase catalytic domain varies between kinases (7). This has led to the view that phosphorylation site recognition occurs through conserved residues flanking the phospho-residue on the substrate. Linear sequence motifs have now been identified for most kinases through a combination of structural analysis and peptide libraries (4, 8). Additionally, these phospho-sites are frequently found in unstructured regions of the substrate protein that may allow for more flexible accommodation in the kinase active site (9, 10). After recognizing the substrate, the catalytic domain then transfers a phosphate group from ATP to the phosphoresidue on the substrate. Phospho-transfer is followed by release of ADP and the phosphorylated substrate to prime the kinase for another catalytic cycle (11).Crystallographic and NMR approaches have provided detailed structural information on the catalytic domains of individual kinases in complex with a variety of nucleotide analogs and inhibitors (6, 12, 13). However, the lack of defined secondary structure surrounding the phospho-motif and the inherent transient nature of the interaction has limited efforts to dissect the different states of the ...

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Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR

Cited by 25 publications

References 55 publications

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

The C-terminal RNA binding motif of HuR is a multi-functional domain leading to HuR oligomerization and binding to U-rich RNA targets

Activation of HuR downstream of p38 MAPK promotes cardiomyocyte hypertrophy

Substrate Affinity Differentially Influences Protein Kinase C Regulation and Inhibitor Potency

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