L-selectin activates the Ras pathway via the tyrosine kinase p56lck

Abstract

Selectins mediate rolling, the initial step of leukocyte adhesion to endothelial cells [Springer, T. A. (1995) Annu. Rev. Physiol. 57, 827-872 and Butcher, E. C. (1991) Cell 67, 1033-1036]. In this study we show that L-selectin triggering of Jurkat cells using different antibodies or glycomimetics resulted in activation of the src-tyrosine kinase p56lck; tyrosine phosphorylation of intracellular proteins, in particular mitogen-activating protein kinase and L-selectin; and association of Grb2/Sos with L-selectin. This association correlated with an activation of p21Ras, mitogen-activating protein kinase, Rac2, and a transient increase of 2-O synthesis. Stimulation of the Ras pathway by L-selectin requires functional p56lck, since p56lck-deficient Jurkat cells (JCaM1.6) do not show tyrosine phosphorylation, association of L-selectin with Grb2/Sos, and activation of Ras upon L-selectin triggering. Transfection of JCaM1.6 cells with p56lck reconstitutes the observed signaling events. Genetic inhibition of Ras or Rac2 prevented Rac2 stimulation and 2-O synthesis, respectively. The specificity and the physiological significance of the observed signaling cascade is indicated by stimulation of L-selectin-transfected P815, L-selectin-positive CEM or peripheral blood lymphocytes resulting in the same activation events as in Jurkat cells. Our results point to a signaling cascade from L-selectin via p56lck, Grb2/Sos, Ras, and Rac2 to 2-O.

Figure 1

l-selectin triggering induces tyrosine phosphorylation dependent on expression of p56^lck. Jurkat, JCaM1.6, JCaM1.6+Lck cells, CEM⁺, CEM⁻, P815.L-Sel, P815.control, or PBL cells were stimulated via l-selectin with Dreg56 or as indicated (A–C). l-selectin triggering induced tyrosine phosphorylation of several proteins in Jurkat, JCaM1.6+Lck, CEM⁺, P815.L-Sel, or PBL cells. No tyrosine phosphorylation was detected in JCaM1.6 cells, CEM⁻, or P815.control cells (A–C). (D) l-selectin stimulation activates p56^lck in Jurkat and JCaM1.6+Lck cells peaking 1 min after stimulation. No activity was seen in JCaM1.6 cells. An aliquot of the IPs was analyzed by Western blotting with anti-p56^lck to test for equal amounts of p56^lck in all IPs (small panels). Cells for non-specific IPs (n.s.) were stimulated with Dreg56 and incubated with an agarose-coupled rabbit antibody. All experiments were repeated at least three times.

Figure 2

(A) l-selectin stimulation induces tyrosine phosphorylation of l-selectin in Jurkat and JCaM1.6+Lck cells only. Cells were stimulated with Dreg56 for the indicated times, l-selectin was immunoprecipitated, and analyzed for tyrosine phosphorylation. Tyrosine phosphorylation of l-selectin was also observed in CEM⁺ (B), P815.L-Sel (B), or PBL cells (C) stimulated with Dreg56 and after activation of Jurkat cells with Fucoidan, sialyl Lewis^X, or Dreg200 (C). Stimulation of Jurkat cells with 25–9, anti-human β₇-integrin (β₇), buffer (b.), CEM⁻, or P815.control cells with Dreg56 did not result in tyrosine phosphorylation (A and B). Non-specific IPs (n.s.) using an irrelevant mouse anti-CD20 antibody showed no l-selectin IP (A). The small blots display aliquots of the IPs blotted with Dreg56 showing similar amounts of l-selectin in all IPs. These blots confirm expression of l-selectin in CEM⁺ and P815.L-Sel cells (B). All experiments were repeated three times.

Figure 3

l-selectin triggering using the indicated stimuli induces tyrosine phosphorylation or activation of MAPK in p56^lck-positive Jurkat, JCaM+Lck, CEM⁺, P815.L-Sel, or in PBL cells. Shown is a phosphotyrosine blot of MAPK IPs (B) or autoradiographies of in vitro kinase assays (B and C). The increase in MAPK activity upon stimulation via CD3 (OKT3, 10 μg/ml) or by PMA (10 ng/ml) served as a positive control (B). No increase of MBP phosphorylation is detected in CEM⁻ and in P815.control cells (C). Non-specific controls (n.s.) represent IPs using a mouse anti-CD20 antibody. Equal amounts of MAPK are shown in the lower panels of all parts by Western blotting of aliquots of the IPs with an anti-MAPK antibody.

Figure 4

l-selectin triggering with Dreg56 or the indicated stimulus activates Ras in Jurkat, JCaM1.6+Lck, CEM⁺, and P815.L-Sel cells. Deficiency of p56^lck in JCaM1.6 cells (A) or of l-selectin (B) prevent Ras activation after l-selectin triggering. Results are representative of three experiments.

Figure 5

(A and B) The GEF Sos, the adapter protein Grb2, and l-selectin associate upon stimulation via l-selectin in Jurkat or JCaM1.6+Lck cells. Association was revealed by co-immunoprecipitations and blotting Sos- or Grb2-IPs with anti-l-selectin or vice versa. No association of Sos, Grb2, and l-selectin was detected in JCaM1.6 cells. All blots were reprobed with the immunoprecipitating antibody (small panels). Non-specific IPs (n.s.) were performed with anti-CD20 as above. The blots are representative of three experiments. (C) Exchange activity in l-selectin IPs increases upon l-selectin triggering. l-selectin IPs were incubated with recombinant GDP. Ras and [³²P-α]-GTP. Increase in binding of [³²P-α]-GTP to Ras reflects exchange activity.

Figure 6

(A) l-selectin triggering stimulates Rac2. Rac2 was immunoprecipitated from [³²P_i]-labeled, Dreg56-treated Jurkat cells and the increase of GTP binding was analyzed by autoradiography. (B) Inhibition of endogenous Ras by N17Ras prevents Rac2 and Ras activation upon l-selectin triggering. Transfection of the vector did not affect l-selectin-induced Rac2 or Ras stimulation. Rac2 or Ras activity in CD20⁺ sorted cells was determined as above. (C) l-selectin triggering leads to synthesis of O₂⁻ in Jurkat and in JCaM1.6+Lck cells. Genetic deficiency of p56^lck, inhibition of Ras with N17Ras or of Rac2 by antisense oligonucleotides prevents O₂⁻ synthesis. Mean values are ± SD.