We have isolated a human cDNA clone encoding a novel protein of 22 kDa that is a human counterpart of the rat oncoprotein PTTG. We show that the corresponding gene (hpttg) is overexpressed in Jurkat cells (a human T lymphoma cell line) and in samples from patients with di erent kinds of hematopoietic malignancies. Analysis of the sequence showed that hPTTG has an aminoterminal basic domain and a carboxyl-terminal acidic domain, and that it is a proline-rich protein with several putative SH3-binding sites. Subcellular fractionation studies show that, although hPTTG is mainly a cytosolic protein, it is partially localized in the nucleus. In addition we demonstrate that the acidic carboxyl-terminal region of hPTTG acts as a transactivation domain when fused to a heterologous DNA binding domain, both in yeast and in mammalian cells.

We recently isolated a cDNA for hpttg, the human homolog of rat pituitary tumor transforming gene. Now we have analysed the expression of hpttg as a function of cell proliferation. hPTTG protein level is up-regulated in rapidly proliferating cells, is down-regulated in response to serum starvation or cell con¯uence, and is regulated in a cell cycle-dependent manner, peaking in mitosis. In addition, we show that hPTTG is phosphorylated during mitosis. Immunodepletion and in vitro phosphorylation experiments, together with the use of a speci®c inhibitor, indicate that Cdc2 is the kinase that phosphorylates hPTTG. These results suggest that hpttg is induced by, and may have a role in, regulatory pathways involved in the control of cell proliferation. Oncogene (2000) 19, 403 ± 409.

We have previously isolated the hpttg proto-oncogene, which is expressed in normal tissues containing proliferating cells and in several kinds of tumors. In fact, expression of hPTTG correlates with cell proliferation in a cell cycle-dependent manner. Recently it was reported that PTTG is a vertebrate analog of the yeast securins Pds1 and Cut2, which are involved in sister chromatid separation. Here we show that hPTTG binds to Ku, the regulatory subunit of the DNA-dependent protein kinase (DNA-PK). hPTTG and Ku associate both in vitro and in vivo and the DNA-PK catalytic subunit phosphorylates hPTTG in vitro. Furthermore, DNA double-strand breaks prevent hPTTG-Ku association and disrupt the hPTTG-Ku complexes, indicating that genome damaging events, which result in the induction of pathways that activate DNA repair mechanisms and halt cell cycle progression, might inhibit hPTTG-Ku interaction in vivo. We propose that hPTTG might connect DNA damage-response pathways with sister chromatid separation, delaying the onset of mitosis while DNA repair occurs.

Grb2 is an adaptor molecule comprising one Src homology (SH) 2 and two SH3 domains. This protein has a natural isoform named Grb3-3 with a deletion within the SH2 domain. Numerous evidence points to a functional connection between SH2-and SH3-containing proteins and molecules implicated in RNA biogenesis. In this context, we have examined the binding of Grb2 and Grb3-3 to heterogeneous nuclear ribonucleoprotein (hnRNP) C. By the use of an in vivo genetic approach and through in vitro experiments, we furnish evidence that both Grb2 and Grb3-3 interact with hnRNP C proteins. Subcellular fractionation studies clearly show that Grb2 is partially localized in the nucleus. In addition, coimmunoprecipitation experiments demonstrate that Grb2⅐hnRNP C complexes exist in intact hematopoietic cells. The carboxyl-terminal SH3 domains of Grb2 and Grb3-3 are primarily responsible for the association with hnRNP C. However, although the proline-rich motif of hnRNP C is involved in the interaction with Grb2, it is not in the binding to Grb3-3. Furthermore, poly(U) RNA inhibits the association of Grb2 with hnRNP C, whereas it enhances the interaction between Grb3-3 and hnRNP C. These findings suggest that the Grb2/Grb3-3-hnRNP C interactions might fulfill different biological functions. Grb2/Ash is the mammalian homolog of Sem-5 and Drk inCaenorhabditis elegans and Drosophila melanogaster, respectively. Grb2 is a ubiquitous 25-kDa protein composed of one Src homology 1 (SH) 2 and two SH3 domains. This adaptor molecule plays an essential role in cell growth and differentiation and, in addition to other functions, connects tyrosine kinase receptors to activation of the Ras pathway. It interacts via its SH3 domains with the proline-rich regions of the mammalian Sos exchange factors. This Grb2⅐Sos complex exists in the cytosol of quiescent cells and upon growth factor stimulation is recruited in an SH2-dependent manner to the plasma membrane, where Sos stimulates nucleotide exchange on Ras (see Ref.

No abstract