J M Westendorf

The MPM2 monoclonal antibody binds to a phospho amino acid-containing epitope present on more than 40 proteins of M-phase eukaryotic cells. We have developed a technique for cloning cDNAs encoding MPM2-reactive phosphoproteins from bacteriophage lambda expression libraries. Proteins from phage plaques were absorbed to nitrocellulose filters, phosphorylated by M-phase kinases, and screened for MPM2 binding. Partial-length cDNAs encoding two MPM2-reactive proteins termed MPM2-reactive phosphoproteins 1 and 2 (MPP1 and MPP2) were isolated. The deduced MPP1 and MPP2 amino acid sequences are not closely related to any previously described proteins. To determine which amino acid stretches contained the MPM2 epitope, sequences from a 15 amino acid peptide expression library were selected for binding to MPM2 after phosphorylation by M-phase kinases. A string of five amino acids was similar among all selected peptides, and the sequence reflecting the most frequent amino acid at each position was Leu-Thr-Pro-Leu-Lys (LTPLK). MPP1 and MPP2 proteins, respectively, contained five and nine sites closely related to LTPLK, including two that were common to both proteins, (F/T)TPLQ and SSP(I/S)D. Peptides containing LTPLK and FTPLQ were strongly phosphorylated by M-phase, but not interphase, cytosolic kinases, and the phosphorylated peptides were bound by MPM2. Thus, we have identified M-phase-specific phosphorylation sites bound by MPM2 and two putative M-phase phosphoproteins containing these sites.

In clams, fertilization is followed by the prominent synthesis of two cyclins, A and B. During the mitotic cell cycles, the two cyclins are accumulated and then destroyed near the end of each metaphase. Newly synthesized cyclin B is complexed with a small set of other proteins, including a kinase that phosphorylates cyclin B in vitro. While both cyclins can act as general inducers of entry into M phase, the two are clearly distinguished by their amino acid sequences (70% nonidentity) and by their different modes of expression in oocytes and during meiosis. In contrast to cyclin A, which is stored solely as maternal mRNA, oocytes contain a stockpile of cyclin B protein, which is stored in large, rapidly sedimenting aggregates. Fertilization results in the release of cyclin B to a more disperse, soluble form. Since the first meiotic division in clams can proceed even when new protein synthesis is blocked, these results strongly suggest it is the fertilization-triggered unmasking of cyclin B protein that drives cells into meiosis I. We propose that the unmasking of maternal cyclin B protein allows it to interact with cdc2 protein kinase, which is also stored in oocytes, and that the formation of this cyclin B/cdc2 complex generates active M phase-promoting factor.

Using an expression cloning technique, we isolated cDNAs for eight M phase phosphoproteins (MPPs 4-11). We then used affinity-purified antibodies to fusion proteins to characterize the intracellular localization and some biochemical properties of these proteins and two others that we identified previously (MPPs 1-2). Each antibody immunoprecipitated one or two protein species of a characteristic size ranging from 17,000 to 220,000 Da. Each MPP, when immunoprecipitated from lysates of M phase cells, was reactive with MPM2, a monoclonal antibody that recognizes a group of related M phase phosphorylation sites, including F-phosphoT-P-L-Q. This reactivity indicated that all the MPPS encoded genuine M phase phosphoproteins. When antibodies to the MPPS were used for immunofluorescence microscopy, each anti-MPP gave a characteristic pattern of localization. In interphase, several of the MPPs were nuclear proteins, whereas others were cytoplasmic or distributed throughout the cell. Three MPPS were strikingly localized to interphase structures: MPP7 to centers of DNA replication, MPP9 to the Golgi complex, and MPP10 to nucleoli. In mitosis, most of the MPPs were distributed throughout the cells. Further studies of the 10 MPPs, most of which are previously undescribed, are expected to provide new understandings of the process of cell division.

Bombesin is a tetradecapeptide which stimulates prolactin secretion in rats and man and in cultures of GH4C1 cells, a clonal strain of rat pituitary tumor cells. We have utilized [125I-Tyr4]bombesin to identify and characterize specific high affinity receptors in GH4C1 cells. Scatchard analysis of equilibrium binding data at 4 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (RT = 3600 +/- 500 sites/cell). The value for the equilibrium dissociation constant (Kd = 1.2 +/- 0.4 nM) agreed closely with the ED50 (0.5 nM) for bombesin stimulation of prolactin release. [125I-Tyr4]Bombesin binding at steady state at 37 degrees C was inhibited by increasing concentrations of unlabeled bombesin in a dose-dependent manner, with an ID50 = 1.4 +/- 0.2 nM. However, binding of [125I-Tyr4] bombesin was not inhibited by 100 nM thyrotropin-releasing hormone, vasoactive intestinal peptide, epidermal growth factor, or somatostatin. Therefore, [125I-Tyr4]bombesin binds to a receptor distinct from the receptors for other peptides which regulate hormone secretion by GH4C1 cells. The analog specificity for high affinity binding showed that the receptors for bombesin recognize the COOH-terminal octapeptide sequence in the molecule. Among five pituitary cell strains tested, two which contained saturable binding sites for [125I-Tyr4]bombesin (GH4C1 and GH3) had previously been shown to respond to bombesin with increased hormone secretion, whereas three which lacked receptors (GC, F4C1, and AtT20/D16v) were unresponsive. Therefore, the [125I-Tyr4]bombesin binding sites appear to be necessary for the biological actions of bombesin. Examination of the processing and metabolism of receptor-bound peptide demonstrated that at 4 degrees C [125I-Tyr4]bombesin binds to receptors on the surface of GH4C1 cells. At 37 degrees C, receptor-bound peptide is rapidly internalized and subsequently degraded in lysosomes. In summary, we have characterized for the first time specific, high affinity pituitary bombesin receptors which are necessary for the biological action of bombesin.