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-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.
Laboratory of Biochemistry, Department of Chemistry, Aristotle University, Thessaloniki, Greece.
Nikolaos
N
Voukkalis
Laboratory of Biochemistry, Department of Chemistry, Aristotle University, Thessaloniki, Greece.
Maria
M
Koutroumani
Institute of Biosciences & Applications, National Centre for Scientific Research "Demokritos", Athens, Greece.
Christoforos
C
Zarkadas
Laboratory of Biochemistry, Department of Chemistry, Aristotle University, Thessaloniki, Greece.
Eleni
E
Nikolakaki
Institute of Biosciences & Applications, National Centre for Scientific Research "Demokritos", Athens, Greece.
Metaxia
M
Vlassi
Laboratory of Biochemistry, Department of Chemistry, Aristotle University, Thessaloniki, Greece.
Thomas
T
Giannakouros
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D000071679:Glycogen Synthase Kinase 3 beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D056004:Molecular Dynamics Simulation; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D000072417:Protein Domains; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097682:Lamin B Receptor
D001120:Arginine; D000071679:Glycogen Synthase Kinase 3 beta; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D018160:Receptors, Cytoplasmic and Nuclear; D012694:Serine; D000097682:Lamin B Receptor; C087728:SRPK1 protein, human