Substrate competition assays.

<p>(A) Phosphorylation of MBP (1.1 μM) and GST-LBRNt(62–92) (0.7 μM) by Akt1 (0.07 μM) in the presence of increasing concentrations of H2B (2.8, 5.6 and 5.6 μM). Upper panel, Coomassie blue staining; lower panel autoradiography. (B) Phosphorylation of H2B (1.4 μM) and GST-LBRNt(62–92) (0.7 μM) by Akt1 (0.07 μM) in the presence of increasing concentrations of R0 peptide (125, 250, 375 and 500 μM). Upper panel, Coomassie blue staining; lower panel autoradiography.</p

Phosphorylation of LBRNt(62–92) and LBRNt(62–92)ΔRS by Akt1.

<p>(A) Amino acid sequence of LBRNt(62–92). The RS domain is underlined and the putative Akt sites (Ser80, Ser82 and Ser84) are marked with an asterisk. (B) Phosphorylation of GST-LBRNt(62–92) and GST-LBRNt(62–92)ΔRS by 0.07 <b>μ</b>M Akt1. The samples were analyzed by SDS-PAGE on 12% gels, stained with Coomassie Blue and autoradiographed.</p

Phosphorylation of LBRNt(62–92) by GST-SRPK1 and Akt1 in the presence of different synthetic peptides.

<p>(A) Amino acid sequences of the peptides used. The relative position of the peptides in LBRNt(62–92) is schematically indicated. 1.95 μM GST-LBRNt(62–92) were incubated with 0.19 μM GST-SRPK1 (B) or 0.07 μM recombinant Akt1 (C) or immunoprecipitated myristoylated HA-Akt1 and FLAG-Akt2 (D) in the presence of 500 μM of each peptide and 25 μM [γ- ³²P]ATP as described under “Materials and Methods”. Samples were subsequently analyzed by SDS-PAGE on a 15% gel and autoradiographed. In (B), (C) and (D) phosphorylation of GST-LBRNt(62–92) is shown in the upper panel and phosphorylation of the peptides is shown in the lower panel.</p

Distance between phosphosites and ATP γ-phosphate in the ternary complexes.

<p>Distance between phosphosites and ATP γ-phosphate in the ternary complexes.</p

Determination of the sites phosphorylated by SRPK1 and Akt1.

<p>Phosphorylation of GST-LBRNt(62–92), GST-LBRNt(62–92)S76G, GST-LBRNt(62–92)S78G, GST-LBRNt(62–92)S80A, GST-LBRNt(62–92)S82A και GST-LBRNt(62–92)S84A by 0.19 μM GST-SRPK1 (left panel) and 0.07 μM Akt1 (right panel). Only the relevant part of the autorad corresponding to the phosphorylated recombinant proteins is shown. Enzyme activity is expressed as a percent of the activity obtained with GST-LBRNt(62–92) which was set to 100 percent. Data represent the means ± SE of three independent experiments. On top of the figure we show a Coomassie Blue staining of the recombinant proteins (1.95 μM of each) used in the phosphorylation assays.</p

SRPK1 and Akt Protein Kinases Phosphorylate the RS Domain of Lamin B Receptor with Distinct Specificity: A Combined Biochemical and <i>In Silico</i> Approach

<div><p>Activated Akt has been previously implicated in acting on RS domain-containing proteins. However, it has been questioned whether its action is direct or it is mediated by co-existing SR kinase activity. To address this issue we studied in detail the phosphorylation of Lamin B Receptor (LBR) by Akt. Using synthetic peptides and a set of recombinant proteins expressing mutants of the LBR RS domain we now demonstrate that while all serines of the RS domain represent more or less equal phosphoacceptor sites for SRPK1, Ser80 and Ser82 are mainly targeted by Akt. 3D-modeling combined with molecular dynamics (MD) simulations show that amongst short, overlapping LBR RS-containing peptides complying with the minimum Akt recognition consensus sequence, only those bearing phosphosites either at Ser80 or Ser82 are able to fit into the active site of Akt, at least as effectively as its known substrate, GSK3-β. Combined our results provide evidence that Akt kinases directly phosphorylate an RS domain-containing protein and that both the residues N-terminal the phosphosite and at position +1 are essential for Akt specificity, with the latter substrate position being compatible with the arginine residue of RS-repeats.</p></div

Atomic details of the interactions of LBR peptides with Akt2 in the simulated ternary complexes.

<p>Details of the interactions of the GSK3-β peptide with Akt2 in the template crystal structure and in the corresponding final 3D-model produced in this study are also shown. The bound peptides are depicted as stick models and are colored according to their atomic B-factors (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154198#sec002" target="_blank">Materials & Methods</a>”): from blue to red for low to high B-factor values, respectively. Critical residues are labeled. Peptide positions are numbered according to the Akt consensus motif. The ANP and ATP molecules are depicted as balls-and-sticks. Magnesium (manganese in the crystal structure) atoms are shown as spheres, in magenta. H-bond interactions are depicted as broken lines. This figure was rendered using PyMOL.</p

3D-modeling of LBR peptides in complex with Akt2.

<p>(A) Sequence alignment of overlapping LBR peptides used for 3D-modeling of Akt2/LBR-peptide complexes and of the GSK3-β peptide. The consensus sequence of the Akt recognition motif is also shown. h stands for large hydrophobic aminoacids. Potential phosphoacceptor serine residues are indicated by an arrow. (B) The known crystal structure of the GSK3-β peptide (in tube) bound to the kinase domain of human Akt2 (in ribbon), used as the modeling template (1O6K) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154198#pone.0154198.ref026" target="_blank">26</a>]. Substrate peptide positions are numbered according to the Akt consensus shown in A. The phosphoacceptor site is indicated by an orange sphere. (C) Results of multiple, independent MD simulations (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154198#pone.0154198.s005" target="_blank">S1 Table</a>) of the LBR peptides shown in A and of the GSK3-β peptide in complex with human Akt2 (as in B). Only the multiple MD models (and the corresponding initial conformations; in yellow) of the bound peptides are shown for clarity. The phosphoacceptor serines are labeled. Molecular model illustrations were rendered using VMD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154198#pone.0154198.ref037" target="_blank">37</a>].</p

Backbone fluctuations of the simulated GSK3-β and LBR peptides bound to Akt2.

<p>Average RMSF values (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154198#sec002" target="_blank">Materials & Methods</a>”) obtained from the corresponding sets of MD simulations of the (left) binary and (right) ternary complexes simulated in this study. Bar lines represent corresponding standard deviations from the mean value within each MD simulation set. Substrate peptide positions are numbered according to the Akt consensus motif (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154198#pone.0154198.g005" target="_blank">Fig 5A</a>).</p