The catalytic activity of the CD45 membrane-proximal phosphatase domain is required for TCR signaling and regulation.

Cell surface expression of CD45, a receptor-like protein tyrosine phosphatase (PTPase), is required for T cell antigen receptor (TCR)-mediated signal transduction. Like the majority of transmembrane PTPases, CD45 contains two cytoplasmic phosphatase domains, whose relative in vivo function is not known. Site-directed mutagenesis of the individual catalytic residues of the two CD45 phosphatase domains indicates that the catalytic activity of the membrane-proximal domain is both necessary and sufficient for restoration of TCR signal transduction in a CD45-deficient cell. The putative catalytic activity of the distal phosphatase domain is not required for proximal TCR-mediated signaling events. Moreover, in the context of a chimeric PTPase receptor, the putative catalytic activity of the distal phosphatase domain is not required for ligand-induced negative regulation of PTPase function. We also demonstrate that the phosphorylation of the C-terminal tyrosine of Lck, a site of negative regulation, is reduced only when CD45 mutants with demonstrable in vitro phosphatase activity are introduced into the CD45-deficient cells. These results demonstrate that the phosphatase activity of CD45 is critical for TCR signaling, and for regulating the levels of C-terminal phosphorylated Lck molecules.     

GRB2 and phospholipase C-gamma 1 associate with a 36- to 38-kilodalton phosphotyrosine protein after T-cell receptor stimulation.

GRB2, a 25-kDa protein comprising a single SH2 domain flanked by two SH3 domains, has been implicated in linking receptor protein tyrosine kinases (PTKs) to the Ras pathway by interacting with the guanine nucleotide exchange protein SOS. Previous studies have demonstrated that GRB2 directly interacts with Shc, a proto-oncogene product that is tyrosine phosphorylated upon receptor and nonreceptor PTK activation. In this report, we detected low levels of tyrosine phosphorylation of Shc and induced association with GRB2 upon T-cell receptor (TCR) stimulation. Instead, a prominent 36- to 38-kDa tyrosine phosphoprotein (pp36-38) associated with the SH2 domain of GRB2 and formed a stable complex with GRB2/SOS upon TCR stimulation. Cellular fractionation studies showed that whereas both GRB2 and SOS partitioned to the soluble and particulate fractions, pp36-38 was present exclusively in the particulate fraction. This phosphoprotein had the same apparent mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the phosphoprotein that associates with phospholipase C-gamma 1 (PLC-gamma 1). Furthermore, following partial immunodepletion of GRB2 and of the associated pp36-38, there was a significant reduction in the amount of the 36-kDa phosphoprotein associated with PLC-gamma 1, suggesting that a trimeric PLC-gamma 1/pp36-38/GRB2 complex could form. In support of this notion, we have also been able to detect low levels of PLC-gamma 1 in GRB2 immunoprecipitates. We suggest that pp36-38 may be a bridging protein, coupling different signalling molecules to cytoplasmic PTKs regulated by the TCR.     

Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.     

Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes

For determination of the extent to which ribosomal DNA (rDNA0 is organized in tandemly repeated arrays, cellular DNA was digested with a restriction enzyme (EcoRV) that does not cut within the single 44-kb rDNA unit, and fragments separated by PFGE were hybridized to specific rDNA probes. A series of bands large enough to contain 15 to more than 30 rDNA repeat units was observed. In YACs containing cloned rDNA, however, such clusters were not observed, presumably because, as shown here for a clone starting with 1.5 tandem repeat units, there is a tendency for repeat units to delete out of the insert. By comparative gel electrophoretic analyses of DNAs from rodent hybrid cells containing singly isolated human chromosomes, most of the bands seen in total human DNA were assigned to at least one of the acrocentric chromosomes. Thus, large characteristic assemblies of DNA containing rDNA and lacking EcoRV sites were stable enough to be conserved in some human/rodent hybrid lines. When further digested with HindIII, which cuts rDNA at several points, the rDNA in each band yielded the expected fragments. If the large species consist completely of clusters of tandemly repeated rDNA units, they account for about half of the total cellular rDNA content estimated by saturation hybridization measurements.     

The Drosophila insulin receptor homolog: a gene essential for embryonic development encodes two receptor isoforms with different signaling potential.

We report the cloning and primary structure of the Drosophila insulin receptor gene (inr), functional expression of the predicted polypeptide, and the isolation of mutations in the inr locus. Our data indicate that the structure and processing of the Drosophila insulin proreceptor are somewhat different from those of the mammalian insulin and IGF 1 receptor precursors. The INR proreceptor (M(r) 280 kDa) is processed proteolytically to generate an insulin-binding alpha subunit (M(r) 120 kDa) and a beta subunit (M(r) 170 kDa) with protein tyrosine kinase domain. The INR beta 170 subunit contains a novel domain at the carboxyterminal side of the tyrosine kinase, in the form of a 60 kDa extension which contains multiple potential tyrosine autophosphorylation sites. This 60 kDa C-terminal domain undergoes cell-specific proteolytic cleavage which leads to the generation of a total of four polypeptides (alpha 120, beta 170, beta 90 and a free 60 kDa C-terminus) from the inr gene. These subunits assemble into mature INR receptors with the structures alpha 2(beta 170)2 or alpha 2(beta 90)2. Mammalian insulin stimulates tyrosine phosphorylation of both types of beta subunits, which in turn allows the beta 170, but not the beta 90 subunit, to bind directly to p85 SH2 domains of PI-3 kinase. It is likely that the two different isoforms of INR have different signaling potentials. Finally, we show that loss of function mutations in the inr gene, induced by either a P-element insertion occurring within the predicted ORF, or by ethylmethane sulfonate treatment, render pleiotropic recessive phenotypes that lead to embryonic lethality. The activity of inr appears to be required in the embryonic epidermis and nervous system among others, since development of the cuticle, as well as the peripheral and central nervous systems are affected by inr mutations.     

Multiple ribosomal RNA cleavage pathways in mammalian cells

The sequence content of mouse L cell pre-rRNA was examined by RNA gel transfer and blot hybridization. Nuclear RNAs were separated by agarose gel electrophoresis, transferred to diazo-paper, and hybridized to twelve different restriction fragments that are complementary to various sections of 45S pre-rRNA. An abundant new 34S pre-rRNA and less abundant new 37S, 26S and 17S pre-rRNAs were detected. The presence of these new pre-rRNAs suggests the existence of at least two new pre-rRNA cleavage pathways. 34S and 26S pre-rRNAs were also detected in HeLa cells suggesting that these new cleavage pathways are characteristic of mammalian cells. Further, an abundant new 12S precursor to 5.8S rRNA was also detected and is common to all the proposed cleavage pathways. The previously identified 45S, 41S, 32S and 20S pre-rRNAs were readily detected and their general structure confirmed. The 20S pre-rRNA is characteristic of the known pathway used by HeLa and other cells, and its presence suggests that growing mouse L cells use this pre-rRNA cleavage pathway. The 36S pre-rRNA characteristic of the previously described mouse L cell cleavage pathway was not detected. In all these cleavage pathways pre-rRNA cleavage sites are apparently identical and occur at or near the termini of the mature 18S, 5.8S and 28S rRNA sequences. The pathways differ only in the temporal order of cleavage at these sites.     

Monoclonal antibody reactive with the human epidermal-growth-factor receptor recognizes the blood-group-A antigen

The hybridoma antibody TL5, which precipitates the EGF receptor from the human epidermoid carcinoma cell line A431, has been shown to recognize the blood-group-A carbohydrate structure. This conclusion has been reached from studies of (a) the binding of the antibody to glycoproteins and haemagglutination of erythrocytes with known blood-group-antigen activities and (b) the inhibition of binding of the antibody to a radiolabelled blood-group-A-active glycoprotein by structurally defined oligosaccharides.     

Substrate phosphorylation specificity of the human c-kit receptor tyrosine kinase.

The chimeric EK-receptor (EK-R), consisting of the epidermal growth factor receptor (EGF-R) extracellular binding domain and p145c-kit cytoplasmic signal-generating sequences, was fully functional in forming high and low affinity EGF binding sites and in ligand-regulated receptor and substrate phosphorylation activities. Relative to EGF-R, EK-R activation stimulated kit-characteristic phosphorylation of human 293 fibroblast substrate polypeptides. Transient coexpression of EK-R with candidate substrates resulted in ligand-induced phosphorylation of phospholipase C gamma and guanosine triphosphatase-activating polypeptide. The RAF-1 serine/threonine kinase was shown to be associated with activated EK-R, but no tyrosine phosphorylation could be detected. The faithfulness of EK-R substrate phosphorylation specificity was confirmed with stem cell factor-stimulated p145c-kit.     

Carboxy-terminal truncations of epidermal growth factor (EGF) receptor affect diverse EGF-induced cellular responses.

The binding of epidermal growth factor (EGF) to its receptor induces tyrosine phosphorylation of phospholipase C gamma (PLC gamma), which appears to be necessary for its activation leading to phosphatidyl inositol (PI) hydrolysis. Moreover, EGF-receptor (EGF-R) activation and autophosphorylation results in binding of PLC gamma to the tyrosine phosphorylated carboxy-terminus of the receptor. To gain further insights into the mechanisms and interactions regulating these processes, we have analyzed transfected NIH-3T3 cells expressing two EGF-R carboxy-terminal deletion mutants (CD63 and CD126) with reduced capacity to stimulate PI hydrolysis, Ca2+ rises, and DNA synthesis. In fact, the CD126 mutant lacking 126 carboxy-terminal amino acids, including four tyrosine autophosphorylation sites, was unable to stimulate PI hydrolysis or Ca2+ rise in response to EGF. Surprisingly, EGF binding to the cell lines expressing CD63 or CD126 mutants was followed by similar stimulation of tyrosine phosphorylation of PLC gamma. Our results suggest that although necessary, tyrosine phosphorylation of PLC gamma may not be sufficient for stimulation and PI hydrolysis. It is clear, however, that the carboxy-terminal region of EGF-R is involved in regulation of interactions with cellular targets and therefore plays a crucial role in postreceptor signaling pathways.     

Fibronectin delays the fusion of L6 myoblasts

The effect of fibronectin on myogenesis has been studied in vitro. The addition of purified fibronectin to the myogenic cell line L₆ blocks fusion and causes an increase in cell number. The effects of fibronectin could be prevented by the immunoprecipitation of fibronectin from solutions using affinity-purified antifibronectin antibodies. Mild trypsinization of the cells (10 μ/ml trypsin for 20 min) which removes surface fibronectin, causes the rate of fusion to increase when the trypsinization is done just before the cells begin to fuse, day 4 (after the plating of the cells), an inhibition when done on one day earlier, day 3, and has no effect when done after the cells have begun to fuse, day 5. By measuring the binding of rhodamine-labeled antifibronectin antibodies to intact cells, it was found that surface fibronectin increased from day 3 to day 4 and then decreased on day 5. The stimulating effect of trypsin on fusion, therefore, corresponds to the day surface fibronectin reaches a peak. Affinity-purified antifibronectin antibodies were also shown to be capable of enhancing fusion. It is concluded from these results that high levels of fibronectin stimulate events which reduce fusion, whereas the removal of surface fibronectin during critical times either stimulates or reduces fusion.