SHORT REPORT Functional association of TGF-b receptor II with cyclin B Jin Hong Liu 1 , Sheng Wei 1 , Pearlie K. Burnette 1 , Ana M Gamero 1 , Michael Hutton 2 and Julie Y Djeu 1 1 Immunology Program, H Lee Mott Cancer Center and Research Institute, Department of Biochemistry and Molecular Biology, University of South Florida, Tampa, Florida 33612; and 2 Mayo Clinic, Jacksonville, Florida, USA Utilizing the cytoplasmic tail of Transforming Growth Factor Receptor Type II (TGFb RII) as bait in a yeast two hybrid system, we have identi®ed human cyclin B2 as a direct physical partner of TGFb RII. Analysis of deletion mutants of glutathione-S-transferase (GST)- cyclin B2 mapped its binding domain for TGFb RII to the C-terminal and revealed a negative regulatory region immediately upstream of the cyclin box. Using recombi- nant proteins, Cdc2 was demonstrated to indirectly interact with TGFb RII via cyclin B2. This interaction was reproduced in THP-1 monocytic cells, where TGFb treatment markedly enhanced the ability of cyclin B2 and, correspondingly, Cdc2 from TGFb-treated THP-l cells, to bind the GST-TGFb RII fusion protein. More importantly, TGFb RII co-precipitated with cyclin B2 in TGFb-treated THP-1 cells. TGFb treatment also caused threonine phosphorylation of Cdc2 in the TGFb RII- cyclin B2-Cdc2 complex in THP1 cells, in parallel with down regulation of Cdc2 function as measured by histone H1 kinase activity. Cyclin B1 had the same capacity to bind TGFb RII and mediate indirect Cdc2 binding. These results suggest an alternative mechanism that cell cycle arrest in the G1/S phase caused by TGFb may, in part, be due to inactivation of cyclin B/Cdc2 kinase, which is needed for entry into the G2/M phase. Keywords: transforming growth factor receptor; cyclin B; cdc-2; cell cycle control Transforming growth factor b (TGFb) is a potent regulator of cell growth and dierentiation. It controls the cell fate by regulating the expression of cyclin- dependent kinases (Cdk), dierentiation factors, and tumor suppressor gene products. TGFb inhibits Mv1Lu lung epithelial cell proliferation by inducing G1 arrest through the inhibition of cyclin D/Cdk4/6, cyclin E/Cdk2, or cyclin A/Cdk2 kinases. TGFb induces down-modulation of Cdk4 synthesis which further leads to inhibition of Cdk2 activation, thus resulting in cell cycle arrest in responsive cells. Constitutive Cdk4 synthesis in these cells leads to TGFb resistance (Ewen et al., 1993; Ko et al., 1993; Satterwhite et al., 1994). TGFb inhibition of cell proliferation is via the induction and activation of the tumor suppressor gene products, p15 and p27 ,that coordinately inhibit Cdk function (Toyoshima and Hunter, 1994; Blain et al., 1997; Reynisdottir and Massague, 1997). In Mv1Lu lung epithelial cells, TGFb could prevent retinoblastoma (Rb) protein phosphor- ylation during G1 phase, and hypophosporylated forms of Rb suppress progression into the S phase (Laiho et al., 1990; Goodrich et al., 1991). By contrast, TGFb stimulates growth in established cell lines of mesenchymal origin and, in C3H mouse ®broblasts paradoxically, it induces cell cycling via activation of cyclin E-Cdk2 kinase (Ravitz et al., 1995). The eect of TGFb is transmitted via a heteromeric complex of type I and type II transmembrane serine/ threonine kinase receptors. The type II receptor (RII) binds TGFb and is constitutively phosphorylated, while TGFb RI cannot bind its ligand without TGFb RII, suggesting that it is probably a downstream substrate of TGFb RII (Wrana et al., 1994; Chen et al., 1995; Chen and Derynck, 1994). However, the signal pathways downstream of these receptors are not completely understood. Several ®ndings suggest that divergent signaling pathways could be mediated with either type I or type II receptor. Speci®c interaction of TGFb RI with the immunophilin FKBP-12 and p21 RAS farnesyltransferase a subunit has been described, suggesting that regulation of the activation of G proteins is one of the mechanisms involved in TGFb- mediated growth inhibitory and dierentiative path- ways (Wang et al., 1994; 1996; Chen et al., 1997). MADR2 is another intermediate protein that directly interacts with TGFb RI, and its phosphorylation is required for nuclear accumulation and initiation of signaling (Macias-Silva et al., 1996). Smad proteins as TGFb signal transducers via TGFb RI have also been reported (Massague et al., 1997; Kretzschmar et al., 1997; Liu et al., 1997). TGFb RII has also been implicated in TGFb- mediated growth arrest, but its mechanism is unclear (Chen et al., 1993, 1995 Derynck, 1994). A WD- domain-containing protein, TRIP-1, which speci®cally associates with TGFb RII in a kinase-dependent manner has been identi®ed. TRIP-1 does not interact with the type II activin receptor or TGFb RI, but is associated with the heterometric TGFb receptor complex (Chen et al., 1995). Other than TRIP, little is known of signal events directly associated with TGFb RII. With this in mind, we utilized a yeast two- hybrid system to identify the direct downstream events from TGFb RII that might be responsible for its eect on cell growth. The cytoplasmic domain of TGFb RII (RIIC) was used as `bait' to screen (Harper et al., 1993; Wang et al., 1994, Chen et al., 1995) a cDNA library from EBV transformed human B cells expressed in the pACT vector (Clontech), to isolate cDNAs for TGFb RII- Correspondence: JY Djeu This work is supported by National Institutes of Health Grant No. RO1-CA63724 Received 18 March 1998; revised 2 July 1998; accepted 3 July 1998 Oncogene (1999) 18, 269 ± 275 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc