CD5 ligation induces low phosphorylation of intracellular substrates and down-modulates ZAP-70 phosphorylation. A, JKHM cells were incubated with anti-CD3 mAb OKT3 (2 μg/ml) or anti-CD5 mAb Y-2/178 (10 μg/ml) for the indicated time points. All stimulations described were induced without the use of cross-linking secondary Abs. Cells were lysed, and global phosphotyrosine content was revealed by Western blotting with the phosphotyrosine-specific mAb 4G10. B, JKHM cells were activated with CD3 or CD5 mAbs for the indicated time points. Subsequently, cells were rapidly collected and resuspended in lysis buffer. Aliquots (90%) of the lysates were subjected to anti-ZAP-70 immunoprecipitation, resolved by 10% SDS-PAGE, and immunoblotted with HRP-labeled anti-phosphotyrosine mAb 410G (B, top panels). C, the rest (10%) of the lysates was directly immunoblotted with anti-phospho-ZAP-70(Tyr⁴⁹³) antibody followed by HRP-conjugated goat anti-rabbit antibody. For loading control, membranes were probed with anti-ZAP-70 antibody (lower panels).

CD5 triggering induces tyrosine phosphorylation of the protein-tyrosine kinase Fyn at the C-terminal inhibitory residue and down-modulates the activity of Fyn. A, JKHM cells were incubated at 37 °C with the anti-CD5 mAb Y-2/178 (10 μg/ml) for the indicated time points or with the isotype control antibody IgG1 (10 μg/ml) for 2 min (corresponding to time point 0 of activation). Subsequently, cells were rapidly collected and resuspended in lysis buffer. Lysates were immunoprecipitated (IP) with polyclonal anti-Lck or anti-Fyn, resolved by 7.5% SDS-PAGE, and blotted onto PVDF filters. Membranes were incubated with HRP-conjugated anti-phosphotyrosine mAb 4G10 (top panels) or with polyclonal anti-Lck or anti-Fyn followed by HRP-labeled goat anti-rabbit antibody (bottom panels). Proteins were visualized by enhanced chemiluminescence. B, JKHM cells were activated with Y-2/178 for the times indicated or incubated with isotype-control antibody (time 0) for 2 min at 37 °C. Subsequently, cells were collected and lysed. Lysates were run in SDS-PAGE and blotted with polyclonal antibodies reacting with phosphorylated Tyr(P)⁵⁰⁵ of Lck or Tyr(P)⁵³¹ of Fyn. Lysates were also blotted with anti-Fyn and anti-Lck polyclonal antibodies, as controls. C, lysates of CD5-stimulated cells for the times indicated were precipitated with anti-pSrc(−) antibodies. After Western blotting, membranes were incubated with either Lck (top) or Fyn (bottom) polyclonal antibodies followed by goat anti-rabbit-HRP incubation and enhanced chemiluminescence.

The kinase activity of Fyn decreases significantly after CD5 stimulation. JKHM cells were activated with Y-2/178 for the indicated time points at 37 °C and lysed in lysis buffer. A, in vitro kinase assays (KA) of Lck and Fyn immunoprecipitates were performed in the presence of [γ-³²P]ATP. Phosphorylated products were separated by 11% SDS-PAGE and visualized by exposure of the dried gels to Eastman Kodak Co. BioMax MR films. IPs were also run in parallel gels and immunoblotted with Lck and Fyn polyclonal antibodies. Changes in the levels of phosphorylation of Lck and Fyn were determined by densitometry. Gels are from one of three independent experiments, and graphs show the mean values and S.D. of Lck and Fyn activities, relative to time 0, from the three experiments. B, kinase assays of Lck and Fyn immunoprecipitates were performed in the presence of [γ-³²P]ATP and an exogenous substrate, a 25-amino acid-long synthetic biotinylated peptide containing the pseudo-immunoreceptor tyrosine-based activation motif of rat CD5. After the kinase reaction, the CD5 peptide was precipitated by streptavidin beads, and radioactive counts associated with the peptide were measured in a Beckman liquid scintillation counter. C, after CD5 stimulation, kinase assays on CD5 immunoprecipitates were also performed. The kinase activity associated with CD5 was detected by autoradiography and measured by densitometry. Values shown in the graph are from three separate experiments. Immunodetection of phosphorylated CD5 in CD5 immunoprecipitates was performed using anti-phosphotyrosine antibodies, confirming that CD5 is tyrosine phosphorylated upon activation. Immunoblotting of total CD5 in the immunoprecipitates is also shown.

Lck association and CD5 tyrosine phosphorylation require sequences of the cytoplasmic tail of CD5. A, shown is a scheme containing the amino acid sequences of the CD5 wild-type molecule and truncation mutants used in this study. Shown in CD5.WT is the amino acid sequence between residues 371 and 471 of the mature protein, with tyrosine residues Tyr³⁷⁸, Tyr⁴²⁹, Tyr⁴⁴¹, and Tyr⁴⁶³ in bold. CD5 truncation mutants (CD5.K384^stop, CD5.E418^stop, and CD5.H449^stop) are named according to the amino acid in which a premature stop codon was introduced. Mutant CD5.H449^stop contains 448 amino acids, 23 less than the full-length protein, and misses only the membrane distal tyrosine residue. Mutants CD5.E418^stop and CD5.K384^stop contain 417 and 383 amino acids, respectively, and both include just the membrane proximal Tyr³⁷⁸. B, expression of CD5 in the indicated cell lines was measured by flow cytometry of cells stained with anti-CD5 mAb followed by FITC-labeled rabbit anti-mouse antibody. Non-transfected 2G5 cells were used as control (gray filling). C, cell lines were surface-biotinylated, lysed in lysis buffer, and CD5 immunoprecipitated with Y-2/178. Immunoprecipitates were either run in SDS-PAGE/Western and subjected to immunoblotting with HRP-conjugated ExtrAvidin (top panel) or subjected to in vitro kinase assays. Half of the CD5 immunoprecipitates in kinase assays were separated by 11% SDS-PAGE and visualized by exposure of Kodak BioMax MR films to the dried gel (2nd panel). The rest of the CD5 immunoprecipitates were denatured in 2% SDS and reprecipitated (Rep) with polyclonal anti-Lck or anti-Fyn antibodies. Lck and Fyn reprecipitates were separated by SDS-PAGE, and signals in dried gels were detected on films (lower panel). To confirm that Fyn can be reprecipitated in the denaturing conditions used, Fyn immunoprecipitated from the different cell lines was subjected to kinase assays, denatured, and reprecipitated using specific anti-Fyn polyclonal antibodies.

The tyrosine kinases responsible for CD5-induced Fyn phosphorylation and for CD5 phosphorylation interact with distinct sequences of the cytoplasmic tail of CD5. A, 2G5/CD5.WT cells were activated with the anti-CD5 mAb Y-2/178 for the indicated time points at 37 °C or incubated for 2 min with isotype-control antibody (time 0). Subsequently, cells were rapidly collected and resuspended in lysis buffer. Lysates were subjected to anti-Fyn immunoprecipitation followed by 7.5% SDS-PAGE and immunoblotting with the HRP-conjugated anti-phosphotyrosine mAb 4G10 or with polyclonal Fyn. Additionally, lysates were run and blotted with polyclonal antibodies reacting with phosphorylated Tyr⁵³¹of Fyn or Tyr(P)⁵⁰⁵ of Lck. Lysates were also blotted with anti-Fyn and anti-Lck polyclonal antibodies, as controls. B, 2G5/CD5.H449^stop and 2G5/CD5.K384^stop cells were activated with CD5 mAb Y-2/178 for the times indicated. After cell lysis, Fyn was immunoprecipitated, and IPs were loaded into SDS-PAGE followed by immunoblotting using 4G10. In parallel with the activation of (mutant CD5-expressing cells shown in stimulations from 0 to 30 min), 2G5/CD5.WT cells were activated under the same conditions (shown for the 2-min time point) as the positive control. 2G5/CD5.WT cells displayed a clear increase in Fyn tyrosine phosphorylation, indicating that cells were properly stimulated. C, JKHM cells were lysed, immunoprecipitated with Csk polyclonal antibodies or CD5 mAb Y-2/178, subjected to SDS-PAGE, and immunoblotted with the indicated antibodies. Arrows show the detection of CD5 and Csk. Empty lanes are indicated by dashes.

CD5 associates with signaling effectors within lipid rafts upon stimulation. A and B, JKHM cells were stimulated with Y-2/178 (anti-CD5) or incubated with isotype-control antibody (NS) for 10 min at 37 °C. Subsequently, cells were collected, washed, and lysed in 1% Triton X-100 lysis buffer. Cell lysates were prepared and subjected to sucrose density centrifugation as described under “Experimental Procedures.” In A, equal volumes of the collected fractions 1–9 were separated by SDS-PAGE and immunoblotted with CD5, Lck, Fyn, or Csk antibodies. Immunodetection of LAT was used as indicative of samples enriched in rafts fractions. Proteins were visualized by enhanced chemiluminescence. In B, raft fractions 1–3 and soluble fractions 7–9 of CD5-activated cells were combined, immunoprecipitated with anti-CD5, and subjected to in vitro kinase assays. Labeled CD5 immune complexes were denatured in 2% SDS and reprecipitated with polyclonal anti-CD5, anti-Lck, anti-Fyn, or anti-Csk antibodies. Products were subjected to SDS-PAGE and visualized by exposure of the dried gels to Kodak BioMax MR films. Rep, reprecipitation.

Upon CD5 ligation CD5 translocates to lipid rafts and associates with signaling effectors. A, PBMC were stimulated with anti-CD5 mAb Y-2/178 or incubated with isotype-control antibody (NS) for 10 min at 37 °C. Total cell lysates were subjected to sucrose density centrifugation as described under “Experimental Procedures.” Fractions recovered were numbered 1–9 from the top of the gradient. An equal volume of each fraction was separated by SDS-PAGE and immunoblotted with either CD5, Lck or Fyn antibodies. The preferential distribution of LAT into the rafts allowed for the identification of raft fractions (not shown). Indicated (framed) fractions were pooled, corresponding to CD5 immune complexes in the “soluble” fraction in non-stimulated PBMC (NS-Sol), CD5 immune complexes in lipid rafts in non-activated PBMC (NS-Raft), CD5 immune complexes in the soluble fraction in CD5-stimulated cells (Act-Sol), and CD5 immune complexes in lipid rafts in CD5-stimulated PBMC (Act-Raft). B, top panel, CD5 was immunoprecipitated from the different pools, run on SDS-PAGE, and immunoblotted using anti-CD5 antibodies. Bottom panel, CD5 immune complexes from the different sets were then subjected to in vitro kinase assays in the presence of [γ-³²P]ATP. After denaturation, samples were run on SDS-PAGE in non-reducing conditions and exposed to autoradiography. C, CD5 complexes were disrupted by heat and reprecipitated with polyclonal CD5, ZAP-70, Lck, Fyn, Csk, PAG, and LAT antibodies. After SDS-PAGE using reducing conditions, radioactive products were visualized by exposure of dried gels to Kodak BioMax MR films. Phosphorylated CD5 in each gel is indicated by an arrow. Numbers on the right represent the molecular mass (MM) of proteins in kDa.

CD5 forms dimers at the cell surface; the cytoplasmic tail of CD5 is not involved in CD5 dimerization or in its translocation to lipid rafts. A, CD2 or CD5 or CD5Ext/CD2Int (a chimera consisting of the extracellular and transmembrane domains of CD5 and the intracellular tail of CD2) was expressed at the surface of 293T cells as BRET pairs (for example by cotransfecting genes encoding CD5_Luc together with CD5_GFP in the same cell or CD2_Luc and CD2_GFP). BRET analysis showed that the CD5 BRET pair (CD5_BP) shows BRET_eff values that can readily be fitted by a dimerization model (34). The CD5Ext/CD2Int chimera follows the same profile as seen for the CD5 wild-type BRET pair, whereas CD2_BP shows low BRET_eff values and independence of the acceptor:donor ratio, characteristics of monomers. B, 2G5/CD5.WT and 2G5/CD5.K384^stop cells were stimulated with anti-CD5 mAbY-2/178 or incubated with isotype-control antibody (NS) for 10 min at 37 °C. Cells were collected, washed, and lysed, and total cell lysates were subjected to sucrose density centrifugation. Equal volumes of each fraction were resolved by SDS-PAGE and blotted onto PVDF filters. Membranes were incubated with CD5 mAb followed by HRP-conjugated rabbit anti-mouse antibody, and proteins were visualized by enhanced chemiluminescence.

CD5 stimulation induces PAG dephosphorylation and PAG-Csk dissociation. A, JKHM cells were incubated with the CD5 mAb Y-2/178 for 2 min or with isotype-control antibody for 2 min (NS). Subsequently, cells were collected, fixed, and permeabilized for 30 min at 4 °C. For flow cytometric analysis, cells were stained with anti-pPAG(Tyr³¹⁷) antibody followed by FITC-labeled anti-rabbit. Histograms are from one of three independent experiments, and graphs shown are the mean values and S.D. of the variation of PAG-Tyr³¹⁷ phosphorylation relative to time 0 from the three experiments. B, detection of pPAG(Tyr³¹⁷) was also performed by immunoblotting. After CD5 activation, cells were lysed, and lysates were run on SDS-PAGE and immunoblotted with antibodies recognizing the Tyr³¹⁷ residue of PAG, and total PAG. The x-fold decrease of PAG(Tyr³¹⁷) phosphorylation was evaluated by densitometric analysis, corrected for the densitometric values of the PAG Western blotting, and is indicated as values under the panels. C, JKHM cells were stimulated with anti-CD5 (Y-2/178) or anti-CD3 (OKT3) for the indicated time periods at 37 °C and lysed in lysis buffer. PAG immunoprecipitates were resolved by 7.5% SDS-PAGE and immunoblotted with anti-Csk polyclonal antibody or with anti-PAG as loading control. The x-fold modulation of PAG-associated Csk was evaluated by densitometric analysis, corrected for the densitometric values of the PAG Western blotting, and is indicated as values under the panels. D, a model for CD5-mediated inhibition of T lymphocyte signaling is shown. CD5 molecules are readily recruited to lipid rafts upon CD5 ligation. CD5 may function as a scaffold bringing together signaling effectors such as Lck, Fyn, and PAG, which can be expressed in different subsets of lipid rafts. Within the complex of pooled rafts, CD5 interactions with PAG and Fyn result in a continuous phosphorylation of the tyrosine residue 317 of PAG catalyzed by Fyn, blocking or slowing down the release of Csk that binds to phosphorylated Tyr³¹⁷ of PAG. Captured or unreleased Csk targets Fyn at the C-terminal inhibitory tyrosine residue, but as Fyn is docked through the SH2 domain and thus kept in an open conformation, is resistant to total inhibition (67). The activity of Fyn is nevertheless decreased and consequently impairs downstream signaling, including ZAP-70 activity. This CD5-controlled modulation of the PAG/Csk/Fyn loop ultimately down-regulates long term T cell responses, such as T cell proliferation.