Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells.
The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.
Rembutsu
Makoto
M
Division of Pathology and Neurosciences, University of Dundee, Ninewells Hospital, Dundee DD1 9SY, Scotland, United Kingdom.
Soutar
Marc P M
MP
Van Aalten
Lidy
L
Gourlay
Robert
R
Hastie
C James
CJ
McLauchlan
Hilary
H
Morrice
Nicholas A
NA
Cole
Adam R
AR
Sutherland
Calum
C
D000595:Amino Acid Sequence; D000818:Animals; D066298:In Vitro Techniques; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats
D011506:Proteins