Abstract: The invention provides methods for isolating a modified peptide from a complex mixture of peptides, the method comprising the steps of: (a) obtaining a proteinaceous preparation from an organism, wherein the preparation comprises modified peptides from two or more different proteins; (b) contacting the preparation with at least one immobilized modification-specific antibody; and (c) isolating at least one modified peptide specifically bound by the immobilized modification-specific antibody in step (b). The method may further comprise the step of (d) characterizing the modified peptide isolated in step (c) by mass spectrometry (MS), tandem mass spectrometry (MS—MS), and/or MS3 analysis, or the step of (e) utilizing a search program to substantially match the spectra obtained for the modified peptide during the characterization of step (d) with the spectra for a known peptide sequence, thereby identifying the parent protein(s) of the modified peptide.

Abstract: The invention discloses two newly-discovered Flt3 phosphorylation sites, tyrosine 589 (Tyr589) and tyrosine 591 (Tyr591) in the intracellular domain, and provides antibodies, both polyclonal and monoclonal, that selectively bind to Flt3 when phosphorylated at these novel sites. Also provided are assays utilizing these reagents, including methods for determining the phosphorylation of Flt3 in a biological sample, selecting a patient suitable for Flt3 inhibitor therapy, profiling Flt3 activation in a test tissue, and identifying a compound that modulates phosphorylation of Flt3 in a test tissue, by using a detectable reagent, such as the disclosed antibodies, that binds to Flt3 when phosphorylated at Tyr589 or Tyr591. The sample or test tissue may be taken from a subject suspected of having cancer, such as acute myelogenous leukemia (AML).

Abstract: A class of motif-specific, context-independent antibodies that bind conserved signal transduction motifs, such as kinase consensus substrate motifs and protein-protein binding motifs, containing one or more modified amino acids, such as a phosphorylated amino acid, is provided. The antibodies bind a plurality of peptides or proteins that contain the modified motif. Context-independent antibodies specific for single modified residues, such as phosphothreonine, are also provided. Methods for producing and using such antibodies are provided.

Abstract: The invention discloses newly-discovered phosphorylation sites in human IRS-1 and IRS-2, serine 1101 (Ser1101) and serine 1149 (Ser1149) respectively, and provides antibodies, both polyclonal and monoclonal, that selectively bind to IRS-1 and/or IRS-2 when phosphorylated at these respective sites, but do not bind to IRS-1 and/or IRS-2 when not phosphorylated at these respective sites. The sites are relevant to insulin-resistance in type 2 diabetes. Also provided are methods for determining the phosphorylation of IRS-1/2 or activity of PKC theta in a biological sample, by using a detectable reagent, such as the disclosed antibodies, that binds to IRS-1/2 only when phosphorylated at Ser1101/Ser1149. Kits comprising the phosphor-IRS-1/2 (Ser1101/1149) antibodies of the invention are also provided.

Abstract: The present invention provides methods for identifying the most relevant signal transduction pathway biomarkers of disease progression, outcome, or therapeutic responsiveness, using phospho-specific antibodies in cellular assays to identify proteins whose activity is correlated to the relevant outcome (e.g. therapeutic responsiveness). The invention also provides a method for utilizing correlated biomarker(s) to predict patient response to a therapeutic composition having efficacy against a disease involving altered signal transduction by employing one or more phospho-specific antibodies to detect activation status of such biomarker(s) in cellular assays. Kits for carrying out the methods of the invention are also provided.

Abstract: The invention provides novel reagents and methods for detecting BCR-ABL or c-Abl kinase activity, and/or Abl signaling pathway activation in a cell or tissue, and discloses novel biomarkers relevant to Abl-mediated disease progression and therapeutic responsiveness, and provides predictive and detection methods based on the same. Phosphorylated BCR-ABL (Tyr245) and/or c-Abl (Tyr245), BCR-ABL (Tyr735) and/or c-Abl (Tyr735), Bcr (Tyr177), CRKL (Tyr207), Gab1 (Tyr627), PYK2 (Tyr402), Tyk2 (Tyr1054/1055), SHP2 (Tyr580), ERK1/2 (Thr202/Tyr204) and MEK1/2 (Ser217/221) have now been identified as relevant biomarkers of c-Abl pathway-mediated disease, and phospho-specific antibodies to these targets are provided. Kits for carrying out the methods of the invention are also provided.

Abstract: The present invention discloses complexes of cellular signaling proteins that interact in vivo with the HIV-encoded auxiliary proteins Nef and Tat to modulate their activity. This complex includes the novel serine/threonine kinase PAK4 and the novel guanine nucleotide exchange factor Cdc42-GEF, which synergize to stimulate Tat transcriptional activity, and the acetyl-transferase Tip60 which modifies Nef. These cellular partners of the HIV auxiliary proteins represent novel targets for HIV therapeutics. The invention provides isolated DNA and vectors encoding PAK4 and Cdc42-GEF, and methods of producing recombinant forms of these proteins. The invention also provides methods for identifying compounds that modulate the activity of HIV-Tat, HIV-Nef or Tip60, and methods for modulating the activity of these enzymes.

Abstract: The invention provides monoclonal antibodies that bind the estrogen receptor &agr; (ER &agr;) when phosphorylated at serine 118 (Ser118) in the N-terminal domain, but do not bind to ER &agr; when not phosphorylated at this site. Also provided are methods for determining the phosphorylation of ER &agr; in a biological sample, profiling ER &agr; activation in a test tissue, and identifying a compound that modulates phosphorylation of ER &agr; in a test tissue, by using the disclosed monoclonal antibodies. The sample or test tissue may be taken from a subject suspected of having cancer, such as breast cancer. Kits comprising the phospho-ER &agr; (Ser118) monoclonal antibodies of the invention are also provided.

Abstract: The invention provides a method for producing antibodies that selectively recognize short, modified amino acid motifs substantially independent of the surrounding amino acid context in which the motif occurs. A novel class of motif-specific, context-independent antibodies is also provided. The invention encompasses modified motifs consisting of single modified amino acids, for example phosphotyrosine or acetylated lysine, as well other modified motifs of multiple amino acids, such as kinase consensus substrate motifs and protein-protein binding motifs relevant to cell signal transduction. Also provided are methods of profiling large and diverse protein populations on a genome-wide basis by utilizing the antibodies of the invention, and methods for the positive identification of cellular phosphoproteins using one or more motif-specific, context-independent antibodies of the invention coupled with protein database searching.

Abstract: The invention provides methods for isolating a modified peptide from a complex mixture of peptides, the method comprising the steps of: (a) obtaining a proteinaceous preparation from an organism, wherein the preparation comprises modified peptides from two or more different proteins; (b) contacting the preparation with at least one immobilized modification-specific antibody; and (c) isolating at least one modified peptide specifically bound by the immobilized modification-specific antibody in step (b). The method may further comprise the step of (d) characterizing the modified peptide isolated in step (c) by mass spectrometry (MS), tandem mass spectrometry (MS-MS), and/or MS3 analysis, or the step of (e) utilizing a search program to substantially match the spectra obtained for the modified peptide during the characterization of step (d) with the spectra for a known peptide sequence, thereby identifying the parent protein(s) of the modified peptide.

Abstract: The present invention is related to a method for producing motif-specific, context-independent antibodies which are specific to at least one modified fixed amino acid residue in the context of variable surrounding amino acid or peptide sequences. The method is particularly useful in producing antibodies which recognize phosphorylated serine, threonine, and tyrosine, or acetylated lysine, as well as other modified amino acid-containing motifs of one or more amino acids.