Activation of signal transduction kinase cascades has been shown to alter androgen receptor (AR) activity. Although it has been suggested that changes in AR phosphorylation might be directly responsible, the basal and regulated phosphorylations of the AR have not been fully determined. We have identified the major sites of AR phosphorylation on ARs expressed in COS-1 cells using a combination of peptide mapping, Edman degradation, and mass spectrometry. We describe the identification of seven AR phosphorylation sites, show that the phosphopeptides seen with exogenously expressed ARs are highly similar to those seen with endogenous ARs in LNCaP cells and show that specific agonists differentially regulate the phosphorylation state of endogenous ARs in LNCaP prostate cancer cells. Treatment of LNCaP cells with the synthetic androgen, R1881, elevates phosphorylation of serines 16, 81, 256, 308, 424, and 650. Ser-94 appears constitutively phosphorylated. Forskolin, epidermal growth factor, and phorbol 12-myristate 13-acetate increase the phosphorylation of Ser-650. The kinetics of phosphorylation of most sites in response to hormone or forskolin is temporally delayed, reaching a maximum at 2 h post-stimulation. The exception is Ser-81, which continues to display increasing phosphorylation at 6 h. These data provide a basis for analyzing mechanisms of crosstalk between growth factor signaling and androgen in prostate development, physiology, and cancer.The steroid hormone receptors are ligand-activated transcription factors. In addition to regulation by steroids, they are also regulated by post-translational modifications generated by signal transduction pathways. Thus, they function not only as transcription factors but also as nodes that integrate multiple extracellular signals. The human progesterone receptor (PR) 1 is phosphorylated on multiple residues; the basal phosphorylation sites, including serines 81, 102, and 162, are rapidly induced in the presence of steroid (1). However, phosphorylation of serines 102, 294, and 345 in response to hormone is temporally delayed, reaching a maximum at 2 h (2). Phosphorylation of PR on Ser-676 in the hinge region has recently been identified (3); the analogous site in the chicken PR is also phosphorylated. When this site is mutated to alanine, subsaturating levels of hormone show severalfold less transcriptional activity compared with wild type (4). At least seven phosphorylation sites have been identified on the glucocorticoid receptor, and the relative level of phosphorylation of these sites appears to be cell cycle-regulated (5-7). Recent evidence indicates that the phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover (8). Growth factors are known to stimulate the ligand-independent activity of the estrogen receptor through the activation of the mitogen-activated protein kinase (MAPK) cascade and the direct phosphorylation of estrogen receptor by MAPK at Ser-118 (9). Hormone binding also regulates the phosphorylation ...

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CD4+ T cells play a critical role in generating and maintaining immune responses against pathogens and alloantigens, and evidence suggests an important role for them in antitumor immunity as well. Although major histocompatibility complex class II–restricted human CD4+ T cells with specific antitumor reactivities have been described, no standard method exists for cloning the recognized tumor-associated antigen (Ag). In this study, biochemical protein purification methods were used in conjunction with novel mass spectrometry sequencing techniques and molecular cloning to isolate a unique melanoma Ag recognized by a CD4+ tumor-infiltrating lymphocyte (TIL) line. The HLA-DRβ1*0101–restricted Ag was determined to be a mutated glycolytic enzyme, triosephosphate isomerase (TPI). A C to T mutation identified by cDNA sequencing caused a Thr to Ile conversion in TPI, which could be detected in a tryptic digest of tumor-derived TPI by mass spectrometry. The Thr to Ile conversion created a neoepitope whose T cell stimulatory activity was enhanced at least 5 logs compared with the wild-type peptide. Analysis of T cell recognition of serially truncated peptides suggested that the mutated amino acid residue was a T cell receptor contact. Defining human tumor Ag recognized by T helper cells may provide important clues to designing more effective immunotherapies for cancer.