Select RSK2 phosphorylated peptides identified by mass spectrometry.

<p>Upper panel shows peptides from samples phosphorylated after incubation with ERK2 and PDK1, but not in nascent protein. Lower panel shows phosphorylated peptides in samples of nascent protein and after incubation with ERK2 and PDK1.</p

Biophysical characterization of RSK2.

<p>(A) Particle size distribution in the sample of RSK2 (0.7 mg/ml) as determined by DLS. Peak 2 accounts for 84% of sample by intensity, has a polydispersity of 24% and corresponds to a particle with an average molecular mass of 144 kDa. (B) Thermal stability assay of RSK2. The minimum (T_m) of the derivative function of fluorescence intensity corresponds to a mid-point of unfolding (denaturation) curve of the protein.</p

Enzymatic activity of RSK2 and its sensitivity to SL0101.

<p><b>(A)</b> Different amounts of RSK2 (1.5–50 ng) were used to phosphorylate S6 peptide (0.2 mg/ml) in the presence of 100 μM ATP for 30 minutes at room temperature in the reaction volume of 5 μl. The percentage of ATP/ADP conversion was estimated from standard curve using ADP Glo assay. <b>(B)</b> The enzymatic activity of RSK2 (5 ng) was assessed at 10 μM of ATP in the presence of indicated concentrations of SL0101 and expressed as a percentage of RSK2 activity in the absence of inhibitor.</p

Phosphorylation of RSK2 <i>in vivo</i>.

<p>Densitometric quantification of RSK2 phosphorylation at Ser227, Thr365, Ser386 and Thr577 after 5 min stimulation with TXA2-analogue U46619 (1 μM) in the presence or absence of the MEK inhibitor U0126 (10 μM) or PDK1 inhibitor GSK2334471 (10μM) normalized to total RSK2 in mouse aortae. Arrows indicate the position of the total RSK2 band in the pSer227 and pThr577 immunoblots. The higher bands likely reflect other RSK isoforms not detected by the specific total RSK antibody, e.g. the anti-pSer227 RSK2 antibody is claimed to be broadly reactive with Ser227 phosphorylated Rsk family proteins and the anti-pThr577 antibody detects RSK1 and RSK 2 and these 2 isoforms differ by 2,099 amino acids in the mouse. <b>(A)</b> Western Blot analysis. <b>(B)</b> Bar-graph summary from three experiments. Rest values are taken as 1. Values shown are means ± S.E.M. * p<0.03, rest compared to U46619 stimulation; # p<0.02, rest compared to MEK inhibitor U0126 pretreatment; ## p<0.02, U46619 stimulation compared to MEK inhibitor U0126; * * p<0.03 U46619 stimulation compared to PDK inhibitor GSK2334471.</p

Time course of RSK2 phosphorylation by ERK2 and PDK1.

<p>Recombinant RSK2 was phosphorylated with the mixture of active PDK1 and ERK2. Equal aliquots of phosphorylation reaction were taken at indicated time points and subjected to immunoblot analysis using antibodies against specific phosphorylated sites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164343#sec002" target="_blank">methods</a>).</p

Structural organization of RSK2 and the canonical scheme of the activating phosphorylation cascade.

<p>Structural organization of RSK2 and the canonical scheme of the activating phosphorylation cascade.</p

Western Blot detection of Ser386 phosphoprotein levels in wild-type and mutant RSK2.

<p>Samples of recombinant RSK2 were assayed for phosphorylation of Ser386 in the nascent form and after incubation with ERK2 and PDK1. The bottom panel shows the total amount of the protein identified by a generic antibody to RSK2 (see text for further details). The splitting of the band in the wild-type sample is occasionally observed likely due to heterogeneity of the sample–not affecting the key conclusion.</p

Purification of MBP-His₆-RSK2.

<p>Proteins were separated by SDS-PAGE and stained using Coomassie Brilliant Blue. Lane 1, pooled fractions after Ni-NTA elution; lane 2, proteins eluted from amylose column; lane 3, proteins pooled fractions after second Ni-NTA elution; 4, proteins after digestion with TEV protease; lane 5, pooled fractions after purification by size exclusion chromatography.</p