Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells

Makoto Rembutsu, Marc P. M. Soutar, Lidy Van Aalten, Robert Gourlay, C. James Hastie, Hilary McLauchlan, Nicholas A. Morrice, Adam R. Cole, Calum Sutherland (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    12 Citations (Scopus)

    Abstract

    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 Pin 1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin 1 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.

    Original languageEnglish
    Pages (from-to)2153-2161
    Number of pages9
    JournalBiochemistry
    Volume47
    Issue number7
    DOIs
    Publication statusPublished - 19 Feb 2008

    Keywords

    • RESPONSE MEDIATOR PROTEINS
    • ALZHEIMERS-DISEASE
    • AXON OUTGROWTH
    • CRMP-2
    • UNC-33
    • PIN1
    • RAT
    • DIHYDROPYRIMIDINASE
    • IDENTIFICATION
    • GUIDANCE

    Cite this

    Rembutsu, Makoto ; Soutar, Marc P. M. ; Van Aalten, Lidy ; Gourlay, Robert ; Hastie, C. James ; McLauchlan, Hilary ; Morrice, Nicholas A. ; Cole, Adam R. ; Sutherland, Calum. / Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells. In: Biochemistry. 2008 ; Vol. 47, No. 7. pp. 2153-2161.
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    abstract = "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 Pin 1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin 1 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.",
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    author = "Makoto Rembutsu and Soutar, {Marc P. M.} and {Van Aalten}, Lidy and Robert Gourlay and Hastie, {C. James} and Hilary McLauchlan and Morrice, {Nicholas A.} and Cole, {Adam R.} and Calum Sutherland",
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    Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells. / Rembutsu, Makoto; Soutar, Marc P. M.; Van Aalten, Lidy; Gourlay, Robert; Hastie, C. James; McLauchlan, Hilary; Morrice, Nicholas A.; Cole, Adam R.; Sutherland, Calum (Lead / Corresponding author).

    In: Biochemistry, Vol. 47, No. 7, 19.02.2008, p. 2153-2161.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells

    AU - Rembutsu, Makoto

    AU - Soutar, Marc P. M.

    AU - Van Aalten, Lidy

    AU - Gourlay, Robert

    AU - Hastie, C. James

    AU - McLauchlan, Hilary

    AU - Morrice, Nicholas A.

    AU - Cole, Adam R.

    AU - Sutherland, Calum

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    AB - 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 Pin 1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin 1 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.

    KW - RESPONSE MEDIATOR PROTEINS

    KW - ALZHEIMERS-DISEASE

    KW - AXON OUTGROWTH

    KW - CRMP-2

    KW - UNC-33

    KW - PIN1

    KW - RAT

    KW - DIHYDROPYRIMIDINASE

    KW - IDENTIFICATION

    KW - GUIDANCE

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    EP - 2161

    JO - Biochemistry

    JF - Biochemistry

    SN - 0006-2960

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