Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells

Abstract

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

Figure 1

Deduced amino acid sequence of rat (R) and human (H) pp36. Asterisks indicate tyrosine residues conserved between rat and human. The oligopeptide sequences obtained from the purified rat pp36 protein were: ILIKPPQITVPR (amino acids 50–61); PATSYPL (amino acids 63–69); and PDLLPIPR (amino acids 79–86). The cDNA sequences are available from EMBL/GenBank/DDBJ under accession numbers AJ223280 and AJ001184.

Figure 2

Expression of pp36 transcripts is limited to T and NK cells. (A) Northern blot analysis using a rat pp36 probe of total RNA from different rat tissues revealed a 1.4-kb transcript in thymus and spleen, but not liver, kidney, testis, brain, muscle, or heart. (B) The probe also detected a 1.4-kb transcript in freshly isolated CD4⁺ and CD8⁺ T cells, Con A lymphoblasts, IL-2–activated NK cells, and in two different NK cell lines (RNK16 and A181) but not in B cells, granulocytes, peritoneal macrophages, or a macrophage line (R2). Probing for β-actin confirmed equal loading of RNA (20 μg/lane; data not shown).

Figure 2

Expression of pp36 transcripts is limited to T and NK cells. (A) Northern blot analysis using a rat pp36 probe of total RNA from different rat tissues revealed a 1.4-kb transcript in thymus and spleen, but not liver, kidney, testis, brain, muscle, or heart. (B) The probe also detected a 1.4-kb transcript in freshly isolated CD4⁺ and CD8⁺ T cells, Con A lymphoblasts, IL-2–activated NK cells, and in two different NK cell lines (RNK16 and A181) but not in B cells, granulocytes, peritoneal macrophages, or a macrophage line (R2). Probing for β-actin confirmed equal loading of RNA (20 μg/lane; data not shown).

Figure 3

A rabbit antiserum to a peptide from the cloned pp36 sequence recognizes a tyrosine-phosphorylated protein of apparent M_r 36 kD that associates with a Grb2-SH2 domain fusion protein. (A) Whole cell lysates of 293T fibroblasts cells and unstimulated rat thymocytes were resolved by SDS-PAGE, transferred to PVDF, and analyzed by immunoblotting with an affinity-purified rabbit antiserum made to a peptide from the cloned rat pp36 sequence. The antiserum detected a protein of apparent M_r 36 kD in thymocytes but not in 293T cells. (B) Lysates from pervanadate-treated (+) or unstimulated (−) rat thymocytes were subjected to immunoprecipitation with anti–PLCγ-1, anti-Grb2, and the anti-pp36 rabbit antiserum. After resolution by SDS-PAGE and transfer to PVDF, the precipitates were analyzed by immunoblotting with an mAb to phosphotyrosine. In the lower panel, parallel blots were probed with anti–PLCγ-1 (146 kD; lanes 1 and 2), anti-Grb2 (24 kD; lanes 3 and 4), and anti-pp36 (36 kD; lanes 5 and 6). (C) Lysates from pervanadate-treated (+) and unstimulated (−) rat thymocytes were subjected to precipitation with Grb2 SH2 domain–GST and Grb2 SH3 domain–GST fusion proteins. The precipitated proteins were analyzed by immunoblotting with the anti-pp36 rabbit antiserum.

Figure 4

HA-pp36 associates with PLCγ-1 and Grb2 in pervanadate-treated 293T cells. (A) 293T cells were transiently transfected with either HA-pp36 sense (+) or antisense (−) expression constructs. Lysates from the transfected cells were subjected to immunoprecipitation with anti-HA mAb, the anti-pp36 rabbit antiserum, or normal rabbit (NR) IgG, and the immunoprecipitates were analyzed by immunoblotting with an mAb to HA. The heavy (H) and light (L) chains of the precipitating HA mAb, revealed by the second step goat anti–mouse IgG antiserum, are indicated. (B) 293T cells were transfected with the HA-pp36 sense expression construct, treated with pervanadate, and solubilized. Immunoprecipitates with anti-Grb2 and anti–PLCγ-1 were analyzed by immunoblotting with anti-HA mAb.