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Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos
Tassula Proikas-Cezanne1, Silvia Stabel, Dieter Riethmacher
1Temple University, Fels Institute for Cancer Research and Molecular Biology, Philadelphia, PA, USA. alexandropolis@yahoo.com
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.
Insights
Researchers identified novel substrates for the Mos protein kinase, including PTP1B and casein, using a constitutively active viral Mos derivative. This discovery aids in understanding Mos kinase activity and developing targeted inhibitors.
Area of Science:
- Molecular Biology
- Oncogene Research
- Protein Kinase Signaling
Background:
- The mos proto-oncogene encodes a serine/threonine-specific protein kinase vital for meiotic cell division.
- A viral Mos derivative, 124-v-Mos, exhibits constitutive kinase activity, enabling substrate screening independent of upstream regulators.
Purpose of the Study:
- To identify novel substrates of the Mos protein kinase using the constitutively active 124-v-Mos.
- To characterize the phosphorylation patterns of identified substrates.
Main Methods:
- Utilized the baculovirus expression system to generate recombinant 124-v-Mos.
- Employed immunocomplex kinase assays to screen for and identify novel Mos substrates.
- Verified Mos-specific phosphorylation using kinase-inactive mutants and analyzed phosphopeptides.
Main Results:
- Identified protein tyrosine phosphatase 1B (PTP1B) and alpha/beta-casein as novel in vitro substrates for 124-v-Mos.
- Demonstrated that 124-v-Mos phosphorylates serine and threonine residues in casein (1:1 ratio) and primarily serine residues in auto-phosphorylation.
- Confirmed Mos-specific phosphorylation of PTP1B and casein using control assays.
Conclusions:
- The identified Mos substrates provide a foundation for determining the Mos-consensus phosphorylation motif.
- This research is crucial for developing specific inhibitors targeting the Mos protein kinase.
