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

Ssn6-Tup1 global transcriptional co-repressor: Role of the N-terminal glutamine-rich region of Ssn6

<div><p>The Ssn6-Tup1 complex is a general transcriptional co-repressor formed by the interaction of Ssn6, a tetratricopeptide repeat (TPR) protein, with the Tup1 repressor. We have previously shown that the N-terminal domain of Ssn6 comprising TPRs 1 to 3 is necessary and sufficient for this interaction and that TPR1 plays critical role. In a subsequent study, we provided evidence that in the absence of Tup1, TPR1 is susceptible to proteolysis and that conformational change(s) accompany the Ssn6-Tup1 complex formation. In this study, we address the question whether the N-terminal non-TPR, glutamine-rich tail of Ssn6 (NTpolyQ), plays any role in the Ssn6/Tup1 association. Our biochemical and yeast-two-hybrid data show that truncation/deletion of the NTpolyQ domain of Ssn6 results in its self association and prevents Tup1 interaction. These results combined with <i>in silico</i> modeling data imply a major role of the NTpolyQ tail of Ssn6 in regulating its interaction with Tup1.</p></div

3D-modeling of Ssn6 fragments.

<p>(A) Modeling of Δ45: (Left) Cartoon representation of the initial 3D-model used for the explicit MD simulation. (Right) Average structure obtained from the last 10ns of the 100 ns MD trajectory, colored according to estimated atomic B-factors: from blue to red for low and high atomic fluctuations, respectively. (B) Modeling of NTpolyQ_TPR1-3: (Left) Cartoon representation of the initial 3D-model of a NTpolyQ_TPRs2.5 fragment used for the REMD simulations. (Right) (a) Average structures obtained from the last 10ns of the solvated 100 ns MD trajectories of the two REMD models (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#sec002" target="_blank">Materials & Methods</a>”). Coloring is according to estimated B-factors, as in A. (b), (c) and (d) Average structures obtained from the last 10ns of the second set of solvated 100 ns MD trajectories of the two NTpolyQ_TPR1-3 models colored according to B-factor values for the entire NTpolyQ_TPR1-3, NTpolyQ and TPR domains, respectively, for clarity. Various domains discussed in the text are labeled. Arrows point to susceptible to proteolysis sites of Ssn6, as described in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref011" target="_blank">11</a>] (also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.g001" target="_blank">Fig 1C</a>).</p

Details of the two final models of the NTpolyQ_TPR1-3 fragment.

<p>(A) The final models (Top: model-1; Bottom: model-2) in cartoon representation. The N11, Qx16 and TPR regions are colored in cyan, yellow and green, respectively. The TPR domain is also depicted as a surface colored according to (B) electrostatic potentials and (C) Ssn6 mutational data; in red/orange: point mutation sites disrupting/affecting Tup1 interaction [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref020" target="_blank">20</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref026" target="_blank">26</a>]. Hydrophobic Ssn6 residues are labeled in B. (D) Cartoon representation of the models with details of the mutations colored in C.</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

Two-hybrid assays for Ssn6-Ssn6 and Ssn6-Tup1 interactions.

<p>Two-hybrid assays for Ssn6-Ssn6 and Ssn6-Tup1 interactions.</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

Prediction of disordered binding regions of Ssn6.

<p>Disorder/binding probability plot for the 540 N-terminal residues of Ssn6 as obtained using the ANCHOR server [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186363#pone.0186363.ref046" target="_blank">46</a>]. Predicted disordered/binding regions are depicted as blue shaded boxes underneath the probability plot.</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

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