Pillars article: the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human T lymphocytes. 1988

Abstract

The CD4 (T4) antigen is a cell-surface glycoprotein that is expressed predominantly on the surface of helper T cells and has been implicated in the regulation of T-cell activation and in the associative recognition of class II antigens of the major histocompatibility complex. In addition, the CD4 antigen appears to serve as a receptor for the human immunodeficiency virus (HIV). An important question has been whether the CD4 receptor is linked to an intracellular mediator that could regulate the activation of the CD4⁺ subset. In this paper, we provide preliminary evidence that the CD4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (PTK) of 55–60 kDa, which is expressed specifically in T cells. The PTK is the human analogue of the murine pp56^LSTRA (pp56^lck) and has significant homology with c-src, c-yes, and other members of the src family. The identification of the PTK associated with CD4 receptor was made by use of an antiserum to a synthetic peptide that was deduced from the DNA sequence of PTK. Two-dimensional nonequilibrium pH gradient gel electrophoresis/NaDodSO₄/PAGE revealed the kinase to focus as a heterogeneous collection of spots in the pH range of 4.0–5.0. Furthermore, in vitro phosphorylation revealed the phosphorylation of two additional polypeptides at 40 and 80 kDa, in addition to the autophosphorylation of the PTK at 55–60 kDa. The potential importance of the association between the CD4 receptor and the PTK of T cells is discussed in relation to T-cell activation and HIV infectivity.

Fig. 1. Phosphorylation by [γ-³²P]ATP of the CD4 antigen from peripheral blood lymphocytes and various cell lines.

A) Lanes; 1, 3, 5, 7, 9, and 11, anti-W6/32 antibody; 2, 4, 6, 8, 10, and 12, anti-CD4 antibody (19thy5D7). Resting cells (lanes 1 and 2); Con A-activated (12 hr) cells (lanes 3 and 4). REX (lanes 5 and 6), HPB-MLT (lanes 7 and 8), U937 (lanes 9 and 10), and Raji (lanes 11 and 12). (B) Phospho amino acid analysis of the individual 55/60-kDa bands precipitated by anli-CD4 antibody.

Fig. 2. NaDodSO₄/PAGE analysis of the association between the CD4 and PTK antigens.

(A) Immunoprecipitates derived from peripheral blood lymphocytes that had been stimulated with Con A for 12 hr were labeled in a kinase assay (as described in Fig. 1). Enolase (1–2 μg; Sigma) was added as substrate to the beads before addition of the reaction mixture. Lanes: 1, anti-PTK antibody; 2, anti-CD4 antibody (12T4D11); 3, anti-CD4 antibody (19thy5D7). (B) Precipitation of the PTK polypeptide from CD4 immunoprecipitation. Anti-CD4 or anti-PTK precipitations were labeled by [γ-³²P]ATP, denatured by boiling in either 1% NaDodSO₄ (lanes 2, 3, 5, and 6) or 2% NaDodSO₄ (lane 4), and diluted to 0.1% NaDodSO₄ by a 1:10 or 1:20 dilution of lysis buffer, The denatured anti-CD4 immunoprecipitates were reprecipitated with the anti-PTK antibody (lanes 3 and 4). Conversely, the denatured anti-PTK immunoprecipitates were reprecipitated with a cocktail of anti-CD4 antibodies (12T4D11, 18T3A9, 19thy5D7) (lane 6). Lanes: 1, anti-CD4 pattern (19thy5D7) prior to denaturation; 2 and 5, anti-lF7 antibody; 3 and 4, anti-PTK antiserum; 6, anti-CD4 antibodies. (C) Phospho amino acid analysis of the polypeptides reprecipitated by the anti-PTK antiserum from immunoprecipitates formed by the anti-CD4 antibody. Polypeptides were eluted and subjected to phospho amino acid analysis as described (Fig. 1).

Fig. 3. Two-dimensional NEPHGE/NaDodSO₄/PAGE of reprecipitated PTK polypeptides.

The anti-PTK antiserum and the anti-CD4 antibody were used to precipitate antigen from Con A-stimulated peripheral blood lymphocytes. Immunoprecipitates were then subjected to labeling with [³²P]ATP, as described in Fig. 1. The labeled polypeptides were eluted from protein A-Sepharose beads by boiling in the presence of 1% (wt/vol) NaDodSO₄ for 5 min and diluting 1:10 in Nonidet P-40 lysis buffer. The anti-PTK antiserum was then used to precipitate antigen from these preparations followed by three washes in lysis buffer and two-dimensional NEPHGE as described (36, 39). (Upper) Anti-PTK precipitated from an anti-PTK immunoprecipitate; (Lower) anti-PTK precipitated from an anti-CD4 immunoprecipitate.