P94fer Facilitates Cellular Recovery of Gamma Irradiated Pre-T Cells
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Oncogene (1997) 14, 2871 ± 2880 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00 p94fer facilitates cellular recovery of gamma irradiated pre-T cells S Halachmy, O Bern, L Schreiber, M Carmel, Y Sharabi, J Shoham and U Nir Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel p94fer is a ubiquitous, nuclear and cytoplasmic tyrosine These three kinases encompass a group of the known, kinase, whose accumulation has been demonstrated in all SH2 containing mammalian nuclear tyrosine kinases mammalian cell lines analysed. In the present work, the (Yates et al., 1995; Wilks and Kurban, 1988; Van Etten p94fer expression pro®le was determined in cell lines et al., 1989; Hao et al., 1989, 1991) which are not src which were not tested before. While being present in related. While p94fer and c-Fes share similar structures, several hematopoietic and non hematopoietic cell lines containing a SH2 domain which is ¯anked by an including thymic stromal cells, the p94fer kinase could not extended N-terminal tail (Wilks and Kurban, 1988; be detected in pre-T and T cell lines. p94fer was also Hao et al., 1989), the c-Abl kinase contains both SH2 absent in pre-B line, but accumulated in these cells upon and SH3 domains and an extended C-terminal portion their induced development to antibody producing cells. (Ren et al., 1994). Several functional motifs have been This is in agreement with the absence of p94fer in primary de®ned in the Abl c-terminus, including a nuclear thymic and splenic T lymphocytes and its induced localization signal (NLS) (Van Etten et al., 1989), accumulation in stimulated B cells. Relatively high DNA binding domain (Kipreos and Wang, 1992), actin p94fer levels were detected in primary thymic and splenic binding domain (Van Etten et al., 1994; McWhirter stromal cells. Ectopic expression of p94fer in pre-T cells and Wang, 1993) and binding sites for the adapter slightly aected their cell cycle pro®le but it did not proteins Crk, Grb-2 and Nck (Ren et al., 1994). The aect their apoptotic death which was induced by N-terminal tail of p94fer exerts a coiled-coil structure ionizing radiation. However, p94fer facilitated dramati- which mediates the interaction of that kinase with cally, the cellular recovery of gamma irradiated pre-T other cellular proteins (Kim and Wang, 1995). Unlike cells which have escaped the apoptotic death. The c-Fes whose expression is con®ned to hematopoietic enhanced recovery of the irradiated, p94fer expressing progenitor cells (MacDonald et al., 1985; Care et al., pre-T cells, resulted most probably from their increased 1996), mature cells of the myeloid lineage (Hanazono survival, rather than from a prominent change in their et al., 1993; Greer et al., 1990; Feldman et al., 1985; proliferation rate. The absence of p94fer from pre-B and Care et al., 1996) and vascular endothelial cells (Greer pre-T cells, may thus contribute to the relative sensitivity et al., 1994), p94fer (Letwin et al., 1988; Hao et al., of these cells to ionizing radiation and to their 1989; Feldman et al., 1986) and c-Abl (Wang and dependence on the functioning of other nuclear tyrosine Baltimore, 1983; Van Etten et al., 1989; Renshaw et al., kinasese. 1988; Muller et al., 1982; Boulter and Wagner, 1988; Bernards et al., 1988) were reported to be ubiquitously Keywords: ionizing-radiation; nuclear; p94fer; T cell; expressed in all tissues analysed. Although diering in tyrosine kinase their tissue distribution, the subcellular localization of c-Fes and p94fer is similar and they accumulate both in the cytoplasm and nucleus of the expressing cells (Yates et al., 1995; Hao et al., 1991). While the cellular Introduction role of the c-Fes nuclear fraction is not understood, its cytoplasmic fraction can promote proliferation of p94fer is an evolutionary conserved (Pawson et al., vascular endothelial cells (Greer et al., 1990) and has 1989), nuclear and cytoplasmic tyrosine kinase (Hao et been implicated in cytokine signal transduction in al., 1991) whose presence has been documented in all hematopoietic cells (Izuhara et al., 1994; Hanazono et mammalian cell lines analysed (Letwin et al., 1988; al., 1993). The cytoplasmic fraction of p94fer interacts Hao et al., 1989; Feldman et al., 1986). A truncated with the catenin-like substrate pp120 in ®broblastic form of p94fer, termed p15ferT, accumulates solely in cells (Kim and Wang, 1995) and its nuclear fraction, meiotic primary spermatocytes (Hazan et al., 1993; was found to associate with the cell chromatin (Hao et Fischman et al., 1990) and is encoded by a testis al., 1991). The kinase activity of the cytoplasmic p94fer, speci®c FER transcript (Keshet et al., 1990; Fischman is elevated in growth factors stimulated ®broblastic cell et al., 1990). p52ferT and p94fer dier in their N-termini lines (Kim and Wang, 1995). However, no direct link but they do share a common SH2 and kinase domains of p94fer to regulation of cell growth has been shown, (Hao et al., 1989; Fischman et al., 1990). The p94fer nor has a p94fer substrate been characterized, thus kinase domain (Letwin et al., 1988; Hao et al., 1989) is leaving the cellular role of p94fer elusive. The 70% and 50% homologous to the kinase domains of ubiquitous expression of p94fer suggest that it may two other known nuclear tyrosine kinases c-Fes (Yates have a function that is fundamental to all cells. The et al., 1995; Wilks and Kurban, 1988; Roebroek et al., cellular levels of p94fer dier however among various 1985) and c-Abl (Van Etten et al., 1989), respectively. cell types (Feldman et al., 1986). While ®broblastic (Hao et al., 1989, 1991), epithelial (Hao et al., 1989, Correspondence: U Nir 1991) and macrophage (U Nir, unpublished data) cell Received 8 August 1996; revised 11 March 1997; accepted lines accumulate high p94fer levels, the expression of 11 March 1997 that tyrosine kinase in some promyelocytic cell lines is Cellular recovery of irradiated pre-T cells SHalachmyet al 2872 relatively low (Hao et al., 1989). In an attempt to et al., 1980). The 70Z/3 dierentiation system was thus elucidate the cellular functions of p94fer, the determina- adopted for checking the dependence of p94fer tion of its accumulation pro®le has been extended to accumulation, on the development of pre-B cells to cell types which have not been analysed before. In this antibody producing B cells. 70Z/3 cells were exposed to work we show that p94fer is not detected in pre-B, pre-T 10 mg/ml LPS and their p94fer accumulation pro®le was and T cell lines and that ectopic expression of p94fer in followed for up to 68 h of LPS treatment. Exposing the pre-T cells, dramatically improved their recovery after 70Z/3 pre-B cells to LPS treatment, led to gradual being exposed to ionizing radiation. The absence of accumulation of p94fer in the maturing cells (Figure 2). p94fer from pre-B and pre-T cells, may thus contribute The kinetic of p94fer accumulation lagged however, to their relative sensitivity to ionizing radiation and to behind the known kinetic of the k light chain the dependence of these cells on the functioning of production. While the k protein level reaches a other ubiquitous nuclear tyrosine kinases, like c-Abl (Tybulewicz et al., 1991; Schwarzberg et al., 1991). a S C2 R1 NIH TEC 127 B Results p94fer is not detected in pre-B, pre-T and T lymphocyte fer cell lines p94 ¨ To extend the previous analysis of the p94fer expression pro®le in various cell types (Letwin et al., 1988; Hao et al., 1989), the relative cellular levels of p94fer were determined in ®ve mouse cell lines which have not been 1234567 analysed before. These included two hematopoietic cell lines: R1 ± a friend erythroleukemia cell line (Marks et al., 1983) and cell line No 127 ± a mouse double positive pre-T thymoma (Irlin and Peled, 1992). The three non hematopoietic cell lines tested were: C2 ± a b 70Z TEC 127 T8 T4 NIH mouse myogenic cell line (Yae et al., 1996), STAC and TEC ± two thymic stromal epithelial cell lines p94fer ¨ (Glimcher et al., 1983). The p94fer levels in these cells were compared to those found in the previously analysed NIH3T3 mouse ®broblastic cell line and the human B cell line DAKIKI (Hao et al., 1989). Whole cell protein extracts prepared from the cultured cells, were fractionated in a SDS ± PAGE, Western blotted and the presence of p94fer was determined with the C1 p94fer antibodies. All the cell lines analysed except for 123456 127 (Figure 1a lane 6), were found to accumulate fer varying levels of the p94fer kinase. The absence of p94fer Figure 1 Accumulation of p94 in mammalian cell lines. (a) Whole cell proteins extracts from: lane 1, STAC cells; lane 2, C2 from the 127 pre-T cells, prompt us to check whether it myogenic cells; lane 3, R1 erythroleukemia cells; lane 4, NIH3T3; is expressed in pre-B and mature T cell lines. The lane 5, TEC cells; lane 6, pre-T 127 cells; and lane 7, DAKIKI B presence of p94fer was thus analysed in three other cells; were resolved in a 9% SDS ± PAGE, Western blotted and reacted with the C1 p94fer antibodies. Arrow on the left indicates mouse lymphocytic cell lines. These included the fer fer + the migration distance of p94 .(b) p94 accumulation in: lane 1, 2B4.11 cell line ± a CD4 T-cell hybridoma (Yang et 70Z/3 cells; lane 2, TEC cells; lane 3, 127 cells; lane 4, CTLL cells; al., 1993), CTLL ± a CD8+ IL-2 dependent cytotoxic T lane 5, 2B4.11 cells; lane 6, NIH3T3.