Murine embryonic ®broblasts lacking TC-PTP display delayed G1 phase through defective NF-kB activation MarõÂa de JesuÂs Ibarra-SaÂnchez 1 , John Wagner 1,2 , Ming-Thong Ong 1,2 , Carmen Lampron 1,2 and Michel L Tremblay* ,1 1 McGill Cancer Centre and Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montreal, Quebec, Canada H3G 1Y6; 2 Kinetek Pharmaceuticals Inc. Vancouver, British Columbia, Canada V6P 6G5 Previous results suggested a potential role for T-cell protein tyrosine phosphatase (TC-PTP) in cell prolifera- tion. However, no conclusive data has supported such a function in the modulation of this process. In order to clarify this issue, we isolated TC-PTP7/7 murine embryonic ®broblasts (MEFs) as well as cell lines to characterize the role of TC-PTP in the control of cell proliferation and cell cycle. Both TC-PTP7/7 primary MEFs and cell lines proliferate slower than TC-PTP +/+ cells. We also demonstrated that TC-PTP7/7 cells have a slow progression through the G1 phase of the cell cycle. Further characterization of the G1 defect indicates that the kinetics of cyclin D1 induction was delayed and that p27 KIP1 remains at higher levels for an extended period of time. Moreover, cells lacking TC- PTP showed a delayed activation of CDK2. This slow progression through the early G1-phase resulted in decreased phosphorylation of the RB protein and subsequent delay into the S phase transition. In contrast, no further defects were detected in other phases of the cell cycle. Survey of the potential signaling pathways leading to this delayed cyclin D1 expression indicated that NF-kB activation was compromised and that IKKb activity was also reduced following PDGF stimulation. Reintroduction of wild-type TC-PTP into the TC- PTP7/7 cells rescued the defective proliferation, cyclin D1 expression, NF-kB activation as well as IkB phosphorylation. Together, these results con®rm that TC-PTP plays a positive role in the progression of early G1 phase of the cell cycle through the NF-kB pathway. Oncogene (2001) 20, 4728 ± 4739. Keywords: T-cell protein tyrosine phosphatase; G1 phase; Cyclin D1; NF-kB; IKK Introduction TC-PTP was one of the ®rst members identi®ed from the protein tyrosine phosphatase (PTP) family. Although originally cloned from a human cDNA T- cell library, it is ubiquitously expressed at all stages of mammalian development and in most adult tissues (Cool et al., 1989, Mosinger et al., 1992, Ibarra- Sa nchez et al., 2000, Tonks and Neel, 2001). TC-PTP contains a nuclear localization signal at its carboxyl terminus that localizes the enzyme to the nucleus (Tillmann et al., 1994; Lorenzen et al., 1995). This exposes the enzyme to a unique set of potential nuclear substrates that may play a role in regulating the cell cycle. Recently, it has been suggested that the rat homologue of TC-PTP associates with chromatin in the nucleus, and phosphorylation by a casein kinase- like enzyme disrupts this association (Nambirajan et al., 2000). Furthermore, it has been shown that TC- PTP associates through its carboxyl terminus with the p97 nuclear import factor (Tiganis et al., 1997). Additional studies have also shown that TC-PTP mRNA levels ¯uctuate during the cell cycle, increasing in G0 and early G1 phase, and decreasing for the rest of the cell cycle (Tillmann et al., 1994; Nambirajan et al., 1995). Finally, overexpression studies suggested that TC-PTP might cause an increase in cell prolifera- tion (Radha et al., 1997). Together these results led us to propose that TC-PTP may play a role in regulation of cell proliferation and cell cycle. Cell cycle progression is controlled by cyclin dependent kinases (CDKs) (Sherr, 1996) that are activated by their association with cyclins and negatively regulated by CDK inhibitors (CKIs) (Sherr and Roberts, 1999; Vidal and Ko, 2000). The control of mammalian cell proliferation by extracellular signals (mitogens) takes place in the mid- to late G1 phase of the cell cycle and is integrated through dierent signaling pathways. The best characterized is the Ras/ Raf kinase/MAPK pathway. Activated forms of Raf can lead to transformation and moderated activity of Raf kinase activity promotes proliferation by inducing cyclin D1 and DNA synthesis (Serrano et al., 1997, Sewing et al., 1997, Woods et al., 1997, Llyod et al., 1997, Kerkho and Rapp, 1998). A second major pathway involves PI3 kinase, and its inactivation has been shown to cause down-regulation of cyclin D1 expression, whereas constitutively active Akt/PKB up- regulates it (Muise-Helmericks et al., 1998; Gille and Downward, 1999). Finally, it has recently been shown that dominant negative forms of the NF-kB transcrip- Oncogene (2001) 20, 4728 ± 4739 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc *Correspondence: ML Tremblay; E-mail: Tremblay@med.mcgill.ca Received 31 October 2000; revised 10 May 2001; accepted 17 May 2001