Phosphorylation‐mediated changes in the electrophoretic mobility of CD5 molecules

Abstract

This work shows that tumor promoter agents (TPA) induce the post-translational modification of the human lymphocyte surface CD5 antigen (Tp67) in several cellular types. Treatment of [32P]orthophosphate-and [35S]cysteine-labeled normal and lymphoblastoid T and B cells with active tumor promoters induced the rapid, transitory and dose-dependent appearance of hyperphosphorylated CD5 forms with higher apparent molecular masses. These changes in the electrophoretic mobility of CD5 molecules were independent of RNA and protein synthesis, as well as of differences in neuraminic acid content. The inhibition of the TPA-mediated changes by protein kinase C inhibitors (staurosporine and 1 -(5-isoquinolylsulfonyl)-2-methylpiperazine) indicated its proteinkinase-C-mediated nature. Phosphatase digestion of CD5 immunoprecipitates reverted the TPA-mediated mobility changes showing its dependence on phosphorylation. Neuraminidase digestion of intact cells revealed that the target of the TPA effects are surface-expressed CD5 molecules. In conclusion, we suggest that the heterogeneity in the electrophoretic mobility induced by TPA could reflect some structural and/or functional differences within CD5 molecules.The CD5 (Tl, Leu-1, Tp67 in human; Ly-1 in mouse) molecule is a 67 kDa surface differentiation glycoprotein present on most peripheral T lymphocytes and thymocytes, as well as on a subset of normal peripheral B lymphocytes [l, 21. Although both the function and the natural ligand of the CD5 antigen still remain unknown, there are functional studies showing that this antigen provides accessory signals necessary for human T lymphocyte activation and proliferation [3]. Perturbation of CD5 by monoclonal antibodies (mAb) has not been observed to activate T cells, however they can increase the CD3iantigen receptor-mediated production of interleukin-2, cell proliferation [4, 51, Ca2+ influx [6] and hydrolysis of inositol phospholipids [7]. Importantly, it has been also shown that although CD5 is not the interleukin-1 (IL-1) receptor itself [8], its expression may regulate responsiveness and binding of 1L-1 [9]. In this respect, IL-1 and CD5-specific mAb had an additive effect on the enhancement of T cell activation, suggesting that the CD5 molecule and the IL-1 receptor are complementary and act through distinct pathways to increase T cell activation [5, 101. However, little is known about the nature of the intracellular mechanism mediating the functional effects of CD5.Protein phosphorylation is the most common form of posttranslational modification used to regulate cellular functions [ll]. Recently, the molecular cloning of the human [12] and Abbreviations. Con A, concanavalin A; IL-1, interleukin-1 ; mAb, monoclonal antibody; PAP, potato acid phosphatase; PBMC, peripheral blood mononuclear cells; PDBU, phorbol 12,13-dibutyrate; PGE2, prostaglandin Ez; PI, isoelectric point; PHA-P, phytohaemagglutinin; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TPA, tumor promoter agent/s; H-7, 1-(5-isoquinolylsulfonyl)-2-meth...

CD5 is phosphorylated on tyrosine after stimulation of the T-cell antigen receptor complex.

“…SA (23,24). Although CD5 has been reported to be constitutively phosphorylated on serine and threonine residues and hyperphosphorylated on serine residues after phorbol ester stimulation (25)(26)(27), to our knowledge, the demonstration of tyrosine phosphorylation of CD5 has not been shown previously. CD5 has a long cytoplasmic domain suggesting a possible role in signal transduction.…”

Role of Two Conserved Cytoplasmic Threonine Residues (T410 and T412) in CD5 Signaling

“…The exact nature of both the kinases and the residues involved in the inducible hyperphosphorylation of CD5 remains to be fully deciphered. Previous data indicate that CD5 is hyperphosphorylated by the protein tyrosine kinases p59 fyn (24), p56 lck (24,28), and ZAP-70 (36), and by the protein serine/threonine kinases PKC (37) and CKII (17). The relevance of the membrane-proximal region of the CD5 cytoplasmic tail for CaMK II␦ association and PKC targeting has been shown (29,31).…”

“…Cell labeling with [ 32 P]orthophosphate was performed as previously reported (37). Briefly, cell transfectants were washed with phosphate-free medium (20 mM HEPES, pH 7.2, 10 mM dextrose, 1 mM CaCl 2 ⅐ 2H 2 O, 5 mM KCl, 150 mM NaCl, 1 mM MgCl 2 ⅐ 6H 2 O, 1 mM MgSO 4 ⅐ 7H 2 O, 20 mM NaHCO 3 , 10% dialyzed FCS) and resuspended to a final density of 1 ϫ 10 7 cells/ml.…”

“…Immunoprecipitation was performed as previously described (37). Briefly, cells were disrupted with lysis buffer (1% Nonidet P-40, 10 mM Tris pH 7.6, 140 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.4 mM sodium orthovanadate, 10 mM iodoacetamide, 5 mM sodium pyrophosphate, 1 mM PMSF, 1 g/ml aprotinin, 1 g/ml pepstatin A, 1 g/ml leupeptin, 1 g/ml chymostatin, and 1 g/ml ␣-1-antitrypsin) for 15 min on ice, and the insoluble fraction was discarded after 15 min of microcentrifugation at 4°C.…”

The Conserved Scavenger Receptor Cysteine-Rich Superfamily in Therapy and Diagnosis

“…Both molecules are constitutively phosphorylated, but lymphocyte activation causes them to become hyperphosphorylated (Cardenas et al, 1990;Lozano et al, 1990a). Although the CD5 signaling pathway is not entirely understood, CD5 cross-linking causes a rapid and transient release of diacylglycerol followed by the activation of PKC and an acid sphingomyelinase (Alberola-Ila et al, 1992; Simarro et al, 1999).…”