Deficient Tyrosine Phosphorylation of c-Cbl and Associated Proteins in Phorbol Ester-resistant EL4 Mouse Thymoma Cells

Abstract

Two tyrosine phosphoproteins in phorbol ester-sensitive EL4 (S-EL4) mouse thymoma cells have been identified as the p120 c-Cbl protooncogene product and the p85 subunit of phosphatidylinositol 3-kinase. Tyrosine phosphorylation of p120 and p85 increased rapidly after phorbol ester stimulation. Phorbol ester-resistant EL4 (R-EL4) cells expressed comparable amounts of c-Cbl and phosphatidylinositol 3-kinase protein but greatly diminished tyrosine phosphorylation. Co-immunoprecipitation experiments revealed complexes of c-Cbl with p85, and of p85 with the tyrosine kinase Lck in phorbol ester-stimulated S-EL4 but not in unstimulated S-EL4 or in R-EL4 cells. In vitro binding of c-Cbl with Lck SH2 or SH3 domains was detected in both S-EL4 and R-EL4 cells, suggesting that c-Cbl, p85, and Lck may form a ternary complex. In vitro kinase assays revealed phosphorylation of p85 by Lck only in phorbol ester-stimulated S-EL4 cells. Collectively, these results suggest that Cbl-p85 and Lck-p85 complexes may form in unstimulated S-EL4 and R-EL4 cells but were not detected due to absence of tyrosine phosphorylation of p85. Greatly decreased tyrosine phosphorylation of c-Cbl and p85 in the complexes may contribute to the failure of R-EL4 cells to respond to phorbol ester.T lymphocyte activation is triggered by interaction between the T cell antigen receptor (TCR) 1 and its cognate antigen. One of the earliest signaling events following TCR stimulation is the rapid increase in tyrosine phosphorylation of a number of proteins (reviewed in Refs. 1 and 2). Unlike growth factor receptors that have intrinsic tyrosine kinase activity (reviewed in Ref. 3), the TCR components transduce their signals through noncovalently associated cytoplasmic tyrosine kinases (1, 2). Three tyrosine kinases, Lck, Fyn, and ZAP-70, have been implicated in the function of the TCR. Identification of substrates for these tyrosine kinases has been an area of intense investigation. Several tyrosine kinase substrates in T cells have been identified in the past few years, including phospholipase C-␥ (4, 5), the guanine nucleotide exchange factor Vav (6 -8), an oligomeric ATPase valosin-containing protein (9, 10), the membrane-cytoskeleton linker protein ezrin (11,12), and the subunit of TCR (13). Recently, c-Cbl, a protooncogene protein of 120 kDa has been identified as another prominent tyrosine kinase substrate in T cells (14). Although it has been demonstrated that c-Cbl becomes rapidly tyrosine-phosphorylated upon TCR stimulation (14) and forms complexes with several signaling molecules (15)(16)(17)(18)(19), the function of c-Cbl in T cells is not clear.T cell activation can be mimicked by the addition of tumorpromoting phorbol esters plus calcium ionophores, suggesting important roles for protein kinase C (PKC) and calcium (20). The role of PKC in T cell activation is further emphasized by studies showing that induction of the transcription factors AP-1, NF-B, and nuclear factor of activated T cell for cytokine gene expression is regulated by PKC (21). PKC is ...

Fyn Associates with Cbl and Phosphorylates Tyrosine 731 in Cbl, A Binding Site for Phosphatidylinositol 3-Kinase

“…These include adapter molecules such as Crk and Grb2 (10, 22, 25, 28 -30, 39, 40), PI 3-kinase (20,21,26,28,29,41,42), and the tyrosine kinases Fyn, Lyn, and Lck (26,27,29,31,36). Differences in proteins associated with Cbl may vary with the type of cell examined, or the receptor system examined.…”

“…Other investigators have demonstrated that Src-like kinases are able to bind to Cbl and that these interactions are mediated by the SH3 domain, the SH2 domain, or both (26,27,29,31,36). It is logical that the SH3 domain of Fyn could bind to Cbl in a phosphotyrosine-independent manner, since SH3 domains recognize proline-rich sequences, and there are several prolinerich regions in Cbl.…”

“…The phosphorylation of Cbl has been observed following activation of numerous receptors, including the T-cell receptor (25)(26)(27)(28), the B-cell receptor (29,30), the Fc receptor (31,32), the epidermal growth factor receptor (20,22,33), erythropoietin receptor (34,35), the IL-3 receptor (18), and the prolactin receptor (PRLR) (19). Numerous signaling molecules have been observed to associate with Cbl including the Src-like kinases Fyn and Lyn (26,27,29,31,36), ZAP-70, and/or Syk (32,37,38), Grb2 (10, 22, 28 -30, 39), Crk (10,25,30,40), Shc (30), and PI 3-kinase (20,21,26,28,29,41,42).…”

Differential expression of FAK and Pyk2 in metastatic and non-metastatic EL4 lymphoma cell lines

“…Responses of “wild-type” (WT) PMA-sensitive EL4 cells to phorbol 12-myristate 13-acetate (PMA) include PKC activation [1–4], tyrosine phosphorylation [5,6], Erk activation [2,4,7,8], adhesion [9], IL-2 production [10–14], and growth arrest [4,11,13,15]. To study the mechanisms by which PMA elicits these responses, we and other groups have characterized PMA-resistant EL4 cells that proliferate in the presence of PMA [4,9,17].…”

RasGRP1 Confers the Phorbol Ester-Sensitive Phenotype to EL4 Lymphoma Cells

“…Responses of sensitive "wild-type" (WT) EL4 cells to phorbol 12-myristate 13-acetate (PMA) include protein kinase C (PKC) activation (Kramer and Sando, 1986;Meier et al, 1991;Baier-Bitterlich et al, 1996;Sansbury et al, 1997), tyrosine phosphorylation (Richardson and Sando, 1995;Luo and Sando, 1997), Erk mitogen-activated protein kinase activation (Meier et al, 1991;Gause et al, 1993;Sansbury et al, 1997), adhesion (Resnick et al, 1997), IL-2 production (Farrar et al, 1980;Sando et al, 1982;Pearlstein et al, 1983;Harrison et al, 1987;Rayter et al, 1992), and growth arrest (Sando et al, 1982;Harrison et al, 1987;Desrivieres et al, 1997;Sansbury et al, 1997). To study the mechanisms by which PMA elicits these responses, we and other investigators have characterized PMA-resistant EL4 cells, which by definition proliferate in the presence of PMA (Resnick et al, 1997;Sansbury et al, 1997;Ku and Meier, 2000).…”