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Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos.

Proikas-Cezanne T, Stabel S, Riethmacher D - BMC Biochem. (2002)

Bottom Line: Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase.Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues.The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Temple University, Fels Institute for Cancer Research and Molecular Biology, Philadelphia, PA, USA. alexandropolis@yahoo.com

ABSTRACT

Background: The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro.

Results: We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B), alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues.

Conclusion: The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

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124-v-Mos phosphorylates α- and β-casein in vitro. Mos kinase assays, in the presence of α- and β-casein, were resolved using 10% SDS-PAGE; the Coomassie stained protein gel shown in 3A, right panel and the corresponding autoradiograph on the left panel. Arrowheads indicate the position of 124-v-Mos, α- and β-casein and the antibody. Using two control immunoprecipitates of Sf9 cells expressing the synthetic kinase-inactive constructs, 124-v-MosK121R or PKCγK380R, Mos-specific β-casein phosphorylation was demonstrated in 3B and 3C: Mos kinase assays were blotted on nylon-membrane, the phospho-β-casein bands (B, arrowhead) excised and 32P-Cerenkov counts recorded (B). Alternatively, the excised phospho-β-casein bands were digested with trypsin and electrophoresed using 16% SDS-PAGE (C). The arrowhead in 3C indicates the tryptic β-casein peptide phosphorylated by wild-type 124-v-Mos only. Further, two-dimensional phosphoamino acid analyses of 124-v-Mos phosphorylated α-casein (D, left panel) and β-casein (D, right panel) were completed, the arrowheads indicating the origins of sample application.
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Figure 3: 124-v-Mos phosphorylates α- and β-casein in vitro. Mos kinase assays, in the presence of α- and β-casein, were resolved using 10% SDS-PAGE; the Coomassie stained protein gel shown in 3A, right panel and the corresponding autoradiograph on the left panel. Arrowheads indicate the position of 124-v-Mos, α- and β-casein and the antibody. Using two control immunoprecipitates of Sf9 cells expressing the synthetic kinase-inactive constructs, 124-v-MosK121R or PKCγK380R, Mos-specific β-casein phosphorylation was demonstrated in 3B and 3C: Mos kinase assays were blotted on nylon-membrane, the phospho-β-casein bands (B, arrowhead) excised and 32P-Cerenkov counts recorded (B). Alternatively, the excised phospho-β-casein bands were digested with trypsin and electrophoresed using 16% SDS-PAGE (C). The arrowhead in 3C indicates the tryptic β-casein peptide phosphorylated by wild-type 124-v-Mos only. Further, two-dimensional phosphoamino acid analyses of 124-v-Mos phosphorylated α-casein (D, left panel) and β-casein (D, right panel) were completed, the arrowheads indicating the origins of sample application.

Mentions: In search of further substrates for the 124-v-Mos protein kinase we tested MBP; histone HI, H2AS, H3; protamine; protaminsulphate; purified PKC-α/-β II/γ and α- and β-casein. With the exception of α- and β-casein (fig. 3A) none of these substrates were phosphorylated by 124-v-Mos (data not shown). The possibility that factors other than 124-v-Mos in the immunoprecipitate might be responsible for the observed casein phosphorylation was eliminated by including a synthetic kinase-inactive construct of 124-v-Mos, 124-v-MosK121R[19], as a control in addition to the Mos-unreleated protein, PKC_K380R. A comparison of background phosphorylation on β-casein in the immunoprecipitates of both controls and 124-v-Mos specific phosphorylation showed that 124-v-Mos phosphorylates β-casein 7fold relative to background (fig. 3B). Critically, a tryptic digest of phosphorylated β-casein revealed that 124-v-Mos phosphorylates a specific tryptic peptide in β-casein which shows no background phosphorylation in either controls (fig. 3C, arrowhead) strongly supporting that 124-v-Mos is able to phosphorylate β-casein. Further, a two-dimensional phosphoamino acid analysis (fig. 3D) showed that 124-v-Mos phosphorylates α- and β-casein on serine and threonine residues at a ratio of 1:1.


Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos.

Proikas-Cezanne T, Stabel S, Riethmacher D - BMC Biochem. (2002)

124-v-Mos phosphorylates α- and β-casein in vitro. Mos kinase assays, in the presence of α- and β-casein, were resolved using 10% SDS-PAGE; the Coomassie stained protein gel shown in 3A, right panel and the corresponding autoradiograph on the left panel. Arrowheads indicate the position of 124-v-Mos, α- and β-casein and the antibody. Using two control immunoprecipitates of Sf9 cells expressing the synthetic kinase-inactive constructs, 124-v-MosK121R or PKCγK380R, Mos-specific β-casein phosphorylation was demonstrated in 3B and 3C: Mos kinase assays were blotted on nylon-membrane, the phospho-β-casein bands (B, arrowhead) excised and 32P-Cerenkov counts recorded (B). Alternatively, the excised phospho-β-casein bands were digested with trypsin and electrophoresed using 16% SDS-PAGE (C). The arrowhead in 3C indicates the tryptic β-casein peptide phosphorylated by wild-type 124-v-Mos only. Further, two-dimensional phosphoamino acid analyses of 124-v-Mos phosphorylated α-casein (D, left panel) and β-casein (D, right panel) were completed, the arrowheads indicating the origins of sample application.
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Figure 3: 124-v-Mos phosphorylates α- and β-casein in vitro. Mos kinase assays, in the presence of α- and β-casein, were resolved using 10% SDS-PAGE; the Coomassie stained protein gel shown in 3A, right panel and the corresponding autoradiograph on the left panel. Arrowheads indicate the position of 124-v-Mos, α- and β-casein and the antibody. Using two control immunoprecipitates of Sf9 cells expressing the synthetic kinase-inactive constructs, 124-v-MosK121R or PKCγK380R, Mos-specific β-casein phosphorylation was demonstrated in 3B and 3C: Mos kinase assays were blotted on nylon-membrane, the phospho-β-casein bands (B, arrowhead) excised and 32P-Cerenkov counts recorded (B). Alternatively, the excised phospho-β-casein bands were digested with trypsin and electrophoresed using 16% SDS-PAGE (C). The arrowhead in 3C indicates the tryptic β-casein peptide phosphorylated by wild-type 124-v-Mos only. Further, two-dimensional phosphoamino acid analyses of 124-v-Mos phosphorylated α-casein (D, left panel) and β-casein (D, right panel) were completed, the arrowheads indicating the origins of sample application.
Mentions: In search of further substrates for the 124-v-Mos protein kinase we tested MBP; histone HI, H2AS, H3; protamine; protaminsulphate; purified PKC-α/-β II/γ and α- and β-casein. With the exception of α- and β-casein (fig. 3A) none of these substrates were phosphorylated by 124-v-Mos (data not shown). The possibility that factors other than 124-v-Mos in the immunoprecipitate might be responsible for the observed casein phosphorylation was eliminated by including a synthetic kinase-inactive construct of 124-v-Mos, 124-v-MosK121R[19], as a control in addition to the Mos-unreleated protein, PKC_K380R. A comparison of background phosphorylation on β-casein in the immunoprecipitates of both controls and 124-v-Mos specific phosphorylation showed that 124-v-Mos phosphorylates β-casein 7fold relative to background (fig. 3B). Critically, a tryptic digest of phosphorylated β-casein revealed that 124-v-Mos phosphorylates a specific tryptic peptide in β-casein which shows no background phosphorylation in either controls (fig. 3C, arrowhead) strongly supporting that 124-v-Mos is able to phosphorylate β-casein. Further, a two-dimensional phosphoamino acid analysis (fig. 3D) showed that 124-v-Mos phosphorylates α- and β-casein on serine and threonine residues at a ratio of 1:1.

Bottom Line: Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase.Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues.The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

View Article: PubMed Central - HTML - PubMed

Affiliation: Temple University, Fels Institute for Cancer Research and Molecular Biology, Philadelphia, PA, USA. alexandropolis@yahoo.com

ABSTRACT

Background: The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro.

Results: We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B), alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues.

Conclusion: The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

Show MeSH