Surface expression of a heterologous phosphatase complements CD45 deficiency in a T cell clone.

Expression of CD45, the major transmembrane protein tyrosine phosphatase expressed on lymphoid cells, is required for optimal T cell receptor (TCR) signal transduction. We and others recently have demonstrated that surface expression of the cytoplasmic domain of CD45 in the absence of its extracellular and transmembrane domains is sufficient to restore TCR-mediated signaling events in CD45-deficient cell lines. Here we demonstrate that a single domain nonreceptor tyrosine phosphatase from yeast expressed as a chimeric protein with the extracellular and transmembrane domains of a major histocompatibility complex class I molecule also is able to restore proximal and distal TCR-mediated signal transduction events in the CD45-deficient T cell line J45.01. Ligation of the TCR on the cell line expressing the yeast phosphatase chimera results in the induction of protein tyrosine kinase activity, soluble inositol phosphate generation, and expression of the CD69 activation antigen. Furthermore, a phosphatase-inactive version of this molecule is unable to restore signal transduction, providing the first formal evidence that plasma membrane associated tyrosine phosphatase activity is required for TCR-mediated signaling.

Rescue of signaling by a chimeric protein containing the cytoplasmic domain of CD45.

Surface expression of the CD45 tyrosine phosphatase is essential for the T cell antigen receptor (TCR) to couple optimally with its second messenger pathways. CD45 may be required to dephosphorylate a TCR-activated protein tyrosine kinase, which then transduces an activation signal from the TCR. A chimeric molecule that contained extracellular and transmembrane sequences from an allele of a major histocompatibility class I molecule and cytoplasmic sequences of CD45 restored TCR signaling in a CD45-deficient mutant T cell line. Thus, expression of the complex extracellular domain of CD45 is not required for the TCR to couple to its signaling machinery.

Tyrosine phosphorylation of Grb2-associated proteins correlates with phospholipase C gamma 1 activation in T cells.

Ligation of the T-cell antigen receptor (TCR) results in the rapid activation of several protein tyrosine kinases, with the subsequent phosphorylation of numerous cellular proteins. We investigated the requirement for tyrosine phosphorylation of proteins which bind the Grb2 SH2 domain in TCR-mediated signal transduction by transfecting the Jurkat T-cell line with a cDNA encoding a chimeric protein designed to dephosphorylate these molecules. Stimulation of the TCR on cells expressing this engineered enzyme fails to result in sustained tyrosine phosphorylation of a 36-kDa protein likely to be the recently cloned pp36/Lnk. Interestingly, TCR ligation of the transfected cells also fails to induce soluble inositol phosphate production and intracellular calcium mobilization, although receptor-mediated tyrosine phosphorylation of phospholipase C gamma 1 still occurs. TCR-mediated Ras and mitogen-activated protein kinase activation remain intact in cells expressing the engineered phosphatase. These data demonstrate that tyrosine phosphorylation of a protein(s) which binds the SH2 domain of Grb2 correlates with phospholipase C gamma 1 activation and suggest that such a phosphoprotein(s) plays a critical role in coupling the TCR with the phosphatidylinositol second-messenger pathway.

Molecular comparison of monocot and dicot U1 and U2 snRNAs.

To elucidate differences between the pre-mRNA splicing components in monocots and dicots, we have cloned and characterized several U1 and U2 snRNA sequence variants expressed in wheat seedling nuclei. Primer extension sequencing on wheat and pea snRNA populations has demonstrated that two 5'-terminal nucleotides found in most other U1 snRNAs are missing/modified in many plant U1 snRNAs. Comparison of the wheat U1 and U2 snRNA variants with their counterparts expressed in pea nuclei has defined regions of structural divergence between monocot and dicot U1 and U2 snRNAs. The U1 and U2 snRNA sequences involved in RNA:RNA interaction with pre-mRNAs are absolutely conserved. Significant differences occur between wheat and pea U1 snRNAs in stem I and II structures implicated in the binding of U1-specific proteins suggesting that the monocot and dicot U1-specific snRNP proteins differ in their binding specificities. Stem III structures, which are required in mammalian systems for splicing complex formation but not for U1-specific protein binding, differ more extensively than stems I, II, or IV. In U2 snRNAs, the sequence differences between these two species are primarily localized in stem III and in stem IV which has been implicated in snRNP protein binding. These differences suggest that monocot and dicot U1 and U2 snRNPs represent distinct entities that may have monocot- and dicot-specific snRNP protein variants associated with each snRNA.

Three domains of SLP-76 are required for its optimal function in a T cell line.

We and others have shown that overexpression of SLP-76 augments TCR-stimulated IL-2 promoter activity in the Jurkat T cell line. In this report we investigate the signaling mechanisms through which SLP-76 mediates its effect on T cell activation. We show that overexpressed SLP-76 acts downstream of TCR-stimulated protein tyrosine kinases, but does not affect calcium signaling. Overexpression of SLP-76 does, however, augment TCR stimulation of both ERK (extracellular signal-regulated kinase) activity and a reporter construct driven by activating protein-1 binding sites. Structure/function analysis reveals that three distinct regions of SLP-76, each important for protein associations, are required for augmentation of TCR-induced nuclear factor-AT activity. These data suggest that SLP-76 functions as an adapter molecule that requires three unique domains to link proximal TCR signals in T cells.

Surface expression of hemopoietic cell phosphatase fails to complement CD45 deficiency and inhibits TCR-mediated signal transduction in a Jurkat T cell clone.

Previous studies have shown that the protein tyrosine phosphatase CD45 is required for initiation of signal transduction through several lymphoid receptors. In contrast, there is increasing evidence that another protein tyrosine phosphatase, hemopoietic cell phosphatase (known as HCP, SHP, PTP1C, SHPTP-1, or PTPN6), is a negative regulator of signaling in hemopoietic cells. To determine the effect of HCP on signal transduction through the TCR, HCP was expressed as a chimeric molecule with extracellular and transmembrane domains of the HLA-A2 molecule (A2/HCP) on wild-type Jurkat T cells and the CD45-deficient variant, J45.01. In this report, we show that expression of A2/HCP, unlike A2 chimeras containing the enzymatic regions of CD45, fails to rescue TCR-mediated signal transduction in J45.01. Furthermore, expression of A2/HCP on wild-type Jurkat T cells results in diminished inositol phosphate production following TCR ligation as well as markedly diminished nuclear factor of activated T cells promoter activity. Surprisingly, however, TCR-mediated tyrosine phosphorylation of phospholipase C gamma 1 remains intact in the Jurkat cells expressing the A2/HCP chimera. These experiments provide further evidence that HCP can serve a negative regulatory role in receptor-mediated signaling in immune cells. Additionally, our studies suggest that surface expression of HCP in T cells may provide a means to identify phosphotyrosine-containing proteins that are required for coupling signaling pathways initiated by ligation of the T cell Ag receptor.

Molecular cloning of SLAP-130, an SLP-76-associated substrate of the T cell antigen receptor-stimulated protein tyrosine kinases.

Previous work has demonstrated that SLP-76, a Grb2-associated tyrosine-phosphorylated protein, augments Interleukin-2 promoter activity when overexpressed in the Jurkat T cell line. This activity requires regions of SLP-76 that mediate protein-protein interactions with other molecules in T cells, suggesting that SLP-76-associated proteins also function to regulate signal transduction. Here we describe the molecular cloning of SLAP-130, a SLP-76-associated phosphoprotein of 130 kDa. We demonstrate that SLAP-130 is hematopoietic cell-specific and associates with the SH2 domain of SLP-76. Additionally, we show that SLAP-130 is a substrate of the T cell antigen receptor-induced protein tyrosine kinases. Interestingly, we find that in contrast to SLP-76, overexpression of SLAP-130 diminishes T cell antigen receptor-induced activation of the interleukin-2 promoter in Jurkat T cells and interferes with the augmentation of interleukin-2 promoter activity seen when SLP-76 is overexpressed in these cells. These data suggest that SLP-76 recruits a negative regulator, SLAP-130, as well as positive regulators of signal transduction in T cells.