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

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 in silico modeling data imply a major role of the NTpolyQ tail of Ssn6 in regulating its interaction with Tup1.

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

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-ß. 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.