Reagents for the detection of protein phosphorylation in T-cell receptor signaling pathways

• US 7,935,790 B2
• Filed: 08/11/2006
• Issued: 05/03/2011
• Est. Priority Date: 10/04/2004
• Status: Active Grant

• Current Assignee: Cell Signaling Technology Incorporated
• Original Assignee: Cell Signaling Technology Incorporated
• Inventors: Rush, John Edward, Lee, Kimberly, Moritz, Albrecht, Polakiewicz, Roberto
• Primary Examiner(s): Goddard; Laura B
• Application Number:
  US11/503,336
• Publication Number:
  US 20070059845A1
• Time in Patent Office: 1,726 Days
• Field of Search: None
• US Class Current: 530/387.1
• CPC Class Codes: C12Q 1/25 involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

The invention discloses 95 novel phosphorylation sites identified in signal transduction proteins and pathways downstream of the T-cell receptor, and provides phosphorylation-site specific antibodies and heavy-isotope labeled peptides (AQUA peptides) for the selective detection and quantification of these phosphorylated sites/proteins, as well as methods of using the reagents for such purpose. Among the phosphorylation sites identified are sites occurring in the following protein types: Actin Binding proteins, Adaptor/Scaffold proteins, Adhesion proteins, Calcium-binding proteins, Cell Cycle Regulation or Channel proteins, Chaperones, Cofactor proteins, Cytoskeletal proteins, DNA Binding proteins, G protein or GTPase Activating proteins, Ligases, Lipid Kinases and Binding proteins, Oxidoreductases, Protein Kinases, Protein Phosphatases, Receptor proteins, RNA Binding proteins, Transcription Factor/Initiation Complex proteins, Transcription Coactivator/Corepressor proteins, Translation Initiation Complex proteins, Ubitquitin Conjugating System proteins, and Vesicle proteins.

Claims

1. An isolated phosphorylation site-specific antibody that specifically binds a human T-cell receptor signaling protein, wherein said signaling protein is cdk6, only when said signaling protein is phosphorylated at the tyrosine at position 13, comprised within the phosphorylatable peptide sequence listed in SEQ ID NO:

60, wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine.

5. An immortalized cell line producing the antibody of any one of claims 1, 2, 3, or 4.

6. The cell line of claim 5, wherein said immortalized cell line is a rabbit hybridoma or a mouse hybridoma.

2. An isolated phosphorylation site-specific antibody that specifically binds a human T-cell receptor signaling protein, wherein said signaling protein is cdk6, only when said signaling protein is phosphorylated at the tyrosine at position 24, comprised within the phosphorylatable peptide sequence listed in SEQ ID NO:

61, wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine.

3. An isolated phosphorylation site-specific antibody that specifically binds a human T-cell receptor signaling protein, wherein said signaling protein is SR-protein-specific kinase, only when said signaling protein is phosphorylated at the tyrosine at position 318, comprised within the phosphorylatable peptide sequence listed in SEQ ID NO:

62, wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine.

4. An isolated phosphorylation site-specific antibody that specifically binds a human T-cell receptor signaling protein, wherein said signaling protein is ZAP70, only when said signaling protein is phosphorylated at the tyrosine at position 248, comprised within the phosphorylatable peptide sequence listed in SEQ ID NO:

63, wherein said antibody does not bind said signaling protein when not phosphorylated at said tyrosine.