T-cell receptor (TCR) cross-linking increases tyrosine phosphorylation of multiple proteins, only a few of which have been identified. One of the most rapidly tyrosine-phosphorylated polypeptides is the 120-kDa product of the proto-oncogene c-cbl, a cytosolic and cytoskeletal protein containing multiple proline-rich motifs that are potential binding sites for proteins containing Src homology 3 (SH3) domains. We report here that in cultured Jurkat T cells, Cbl is coprecipitated with antibody against the adapter protein Grb2. Upon activation of Jurkat T cells via the TCR-CD3 complex, we find that high-affinity binding of Cbl requires the N-terminal SH3 domain of GST-Grb2 fusion protein but after cross-linking of the TCR-CD3 and CD4 receptors, Cbl binds equally to its SH2 domain. Grb2 antisera also precipitated p85 from serum-starved cells, while TCR activation increased p85 and tyrosine-phosphorylated Cbl but not Cbl protein in Grb2 immunocomplexes. Phosphatidylinositol (PI) 3-kinase activity was immunoprecipitated from serum-starved cells with Cbl and to a lesser extent with Grb2 antisera, and TCR cross-linking increased this activity severalfold. The PI 3-kinase activity associated with Cbl amounted to 5 to 10% of the total cellular activity that could be precipitated by p85 antisera. The Ras exchange factor Son-of-sevenless 1 (Sos-1) was not found in anti-Cbl immunoprecipitates from activated cells, and Cbl was not detectable in anti-Sos-1 precipitates, supporting the likelihood that Sos-Grb2 and Cbl-Grb2 are present as distinct complexes. Taken together, these data suggest that Cbl function in Jurkat T cells involves its constitutive association with Grb2 and its recruitment of PI 3-kinase in response to TCR activation.

The effects of starvation, glucose refeeding, dibutyryl cAMP, and dexamethasone on expression of the gene for phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from rat liver cytosol was studied by using a cloned cDNA probe. The rate of transcription of the gene for phosphoenolpyruvate carboxykinase in hepatic nuclei isolated from starved rats decreased rapidly after refeeding with glucose. Administration of dibutyryl cAMP to glucose-refed animals increased the rate ofphosphoenolpyruvate carboxykinase gene transcription seven-fold within 20 min. Phosphoenolpyruvate carboxykinase mRNA in the cytosol is 2.8 kldobases long whereas liver nuclei contain four precursor RNA species that are up to 6.5 kilobases long. Feeding glucose to starved rats rapidly decreased the sequence abundance of enzyme mRNA in both nuclei and cytosol. However, the decrease in cytosolic phosphoenolpyruvate carboxykinase mRNA was preceded by a transient increase in enzyme mRNA over the first 20 min after glucose refeeding. Administration of dibutyryl cAMP to glucoserefed starved animals increased the concentration of the nuclear RNA precursors of phosphoenolpyruvate carboxykdnase five-to eight-fold within 30 min and induced the mRNA for the cytosolic enzyme over a period of 60 min. We conclude that cAMP induces phosphoenolpyruvate carboxykdnase mRNA by increasing the rate of gene transcription.The mechanism by which cAMP controls the synthesis rate of hepatic enzymes such as phosphoenolpyruvate (P-enolpyruvate) carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] has not been directly determined. N6,02'-Dibutyryladenosine 3':5'-cyclic monophosphate (Bt2cAMP) administration to a glucose-fed rat rapidly increases the levels of cytosolic P-enolpyruvate carboxykinase mRNA (1-3) and this effect can be blocked by simultaneous administration of cordycepin or actinomycin D (1), suggesting that cAMP acts at the level of gene transcription. However, there are also a number of reports indicating that cAMP regulates the translation of both P-enolpyruvate carboxykinase (4) and tyrosine aminotransferase. (5, 6) mRNA. The availability of a cDNA probe to P-enolpyruvate carboxykinase from rat liver cytosol (2) provides a direct method to determine the mechanism of action of cAMP on expression of the gene for this enzyme. In this paper, we present evidence that administration of Bt2cAMP induces a rapid increase in the rate oftranscription of the P-enolpyruvate carboxykinase gene as measured in rat liver nuclei in vitro.MATERIALS AND METHODS Chemicals. The following nucleotides and reagents were purchased from New England Nuclear or Amersham Searle:[3H]UTP (52 Ci/mmok, 1 Ci = 3.7 X 1010 becquerels), [a-32P]dCTP (400-600 Ci/mmol), [a-32P]UTP (410 Ci/mmol), and DNA polymerase I. Nitrocellulose (BA-85) was from Schleicher & Schuell; nucleotides and dextran sulfate were from Boehringer Mannheim and Pharmacia, respectively. Guanidinium thiocyanate was purchased fr...

The gene for cytosolic phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32] from the rat was isolated from a recombinant library containing the rat genome in phage A Charon 4A. The isolated clone, APCK1, contains the complete gene for phosphoenolpyruvate carboxykinase and -7 kilobases (kb) of flanking sequence at the 5' end and 1 kb at the 3' terminus. Restriction endonuclease mapping, R-loop mapping, and partial DNA sequence assay indicate that the gene is -6.0 kb in length (coding for a mRNA of 2.8 kb) and contains eight introns. Southern blotting of rat DNA digested with various restriction enzymes shows a pattern predicted from the restriction map of APCK1. A control region at the 5' end of the gene contained in a 1.2-kb restriction fragment was isolated and subcloned into pBR322. This segment of the gene contains the usual transcription start sequences and a 24-base sequence virtually identical to the sequence found in the 5'-flanking region of the human proopiomelonocortin gene, which is known to be regulated by glucocorticoids. The 1.2-kb fragment of the phosphoenolpyruvate carboxykinase gene can be transcribed into a unique RNA fragment of predicted size by an in vitro transcription assay.Phosphoenolpyruvate carboxykinase (GTP) [GTP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32; P-enolpyruvate carboxykinase] is a key regulatory enzyme in hepatic gluconeogenesis, and its synthesis rate is controlled by a number of hormones including glucagon (acting through cAMP) (1, 2), cAMP (1), insulin (2, 3), epinephrine (4), glucocorticoids (5), and thyroid hormone (6). Of particular importance are: cAMP, which can induce the synthesis rate of P-enolpyruvate carboxykinase from rat liver cytosol 8-fold in 90 min (7); insulin, which causes an equally rapid deinduction of enzyme synthesis when administered to diabetic animals (2); and glucocorticoids, which also stimulate the synthesis of the enzyme (5). These rapid changes in the synthesis of hepatic P-enolpyruvate carboxykinase are accompanied by equally rapid changes in the sequence abundance of the mRNA coding for the enzyme (8,9

Phosphatidylinositol-3 kinase (PI-3 kinase) has been implicated in cellular events such as mitogenic signaling, actin organization, and receptor sorting. The p85 subunit of PI-3 kinase contains multiple domains capable of protein-protein interactions that may contribute to mediate the multiple physiological functions of this enzyme. Here, we demonstrate that antibodies raised against the p85 subunit of PI-3 kinase immunoprecipitate a single tyrosine-phosphorylated protein of 120 kDa (pp120) from lysates of activated Jurkat T cells and A20 B cells. This protein is the only significant phosphotyrosine-containing protein in p85 immunoprecipitates from these cells, and it cannot be detected in immunoprecipitates of other signaling proteins such as PLC gamma. Furthermore, antibodies specific for the beta isoform of p85 but not antibodies specific for the alpha isoform immunoprecipitate this tyrosine-phosphorylated protein. pp120 completely comigrates with the proto-oncogene c-cbl, which is a 120 kDa protein product abundant in lymphoid cells. Furthermore, immunoblots of p85 immunoprecipitates using antibodies raised against c-cbl detect a band at exactly the position of pp120. In addition, p85 can be detected in immunoblots of c-cbl immunoprecipitates. Thus, pp120 appears to correspond to c-cbl. A direct association between c-cbl and p85 can be observed in vitro using a fusion protein comprising the Src homology 2 (SH2) domains of p85, and this binding is abolished by phenyl phosphate, suggesting that the interaction is mediated through phosphotyrosine-SH2 domain interactions. Thus, these results show important functional differences between the alpha and beta isoforms of p85 in vivo and point to c-cbl as a potentially important mediator of some of the functions of PI-3 kinase in intact cells.

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