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Signal transduction by CXC chemokine receptor 4. Stromal cell-derived factor 1 stimulates prolonged protein kinase B and extracellular signal-regulated kinase 2 activation in T lymphocytes.

Tilton B, Ho L, Oberlin E, Loetscher P, Baleux F, Clark-Lewis I, Thelen M - J. Exp. Med. (2000)

Bottom Line: In addition, restimulation yields >60% of initial protein kinase B activity, indicating that the remaining receptors are not desensitized.Furthermore, activation is prolonged by inhibiting SDF-1 degradation.The sustained activation of cell survival and mitogenic pathways may account for the unique role of SDF-1 and CXCR4 in embryogenesis and lymphopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Theodor Kocher-Institute, University of Bern, CH-3000 Bern 9, Switzerland.

ABSTRACT
We report that stromal cell-derived factor (SDF)-1 has the remarkable capacity to induce sustained signaling through CXC chemokine receptor 4 (CXCR4). In contrast to other chemokines, such as monocyte chemotactic protein 1 (CC chemokine receptor 2 [CCR2]), macrophage inflammatory protein 1beta (CCR5), liver and activation-regulated chemokine (LARC [CCR6]), Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC [CCR7]), and IP10 (CXCR3), SDF-1 stimulates the prolonged activation of protein kinase B and extracellular signal-regulated kinase (ERK)-2. Activation of protein kinase B is reversed by displacement of SDF-1 from CXCR4 or inhibition of phosphatidylinositol 3-kinase. Although increasing concentrations of SDF-1 enhance CXCR4 internalization, kinase activation is prolonged. In addition, restimulation yields >60% of initial protein kinase B activity, indicating that the remaining receptors are not desensitized. Furthermore, activation is prolonged by inhibiting SDF-1 degradation. The sustained activation of cell survival and mitogenic pathways may account for the unique role of SDF-1 and CXCR4 in embryogenesis and lymphopoiesis.

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SDF-1 concentration dependence of prolonged protein kinase B and ERK-2 activation. (A) Western blot analysis of protein kinase B. T cells were stimulated with 10, 30, 100 nM or 1 μM SDF-1 for the indicated times. Activated (pSer473 PKB) and total protein kinase B (PKB) were determined as in the legend to Fig. 1 A. (B) Protein kinase B activity in immunoprecipitates from lysates of cells stimulated with the indicated concentrations of SDF-1 for either 1 min (•) or 20 min (○). Results are expressed as fold activation. (C) Western blot analysis of ERK-2. T cells were stimulated with 10, 100 nM and 1 μM SDF-1 for the indicated times. Activated ERK-2 (pERK-2) corresponds to the band with retarded electrophoretic mobility. Results are representative for at least three independent determinations.
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Figure 4: SDF-1 concentration dependence of prolonged protein kinase B and ERK-2 activation. (A) Western blot analysis of protein kinase B. T cells were stimulated with 10, 30, 100 nM or 1 μM SDF-1 for the indicated times. Activated (pSer473 PKB) and total protein kinase B (PKB) were determined as in the legend to Fig. 1 A. (B) Protein kinase B activity in immunoprecipitates from lysates of cells stimulated with the indicated concentrations of SDF-1 for either 1 min (•) or 20 min (○). Results are expressed as fold activation. (C) Western blot analysis of ERK-2. T cells were stimulated with 10, 100 nM and 1 μM SDF-1 for the indicated times. Activated ERK-2 (pERK-2) corresponds to the band with retarded electrophoretic mobility. Results are representative for at least three independent determinations.

Mentions: The prolonged activation of protein kinase B elicited by SDF-1 was unexpected and appeared to be a distinctive characteristic of the Gi protein–coupled receptor CXCR4. Therefore, we investigated whether SDF-1 causes protracted activation of other intracellular signal transduction pathways. Previous studies demonstrated that the MAP kinase kinase cascade leading to the activation of ERK-2 is regulated independently of protein kinase B 28, and chemokines were shown to stimulate ERK-2 activity in Jurkat cells 12. Using the same panel of chemokines, we compared the activation of ERK-2 in T cells. Fig. 1 D represents a Western blot analysis of ERK-2 activation performed with the same lysates as used in Fig. 1 A. Stimulation of ERK-2 results in the retarded electrophoretic mobility of the threonine- and tyrosine-phosphorylated enzyme that is detected by a specific antibody reacting with both resting and activated enzyme. Fig. 1 D shows that the most prominent and long-lasting effect was again obtained with SDF-1. With 100 nM SDF-1, phosphorylated ERK-2 was detectable for up to 90 min (see Fig. 4). The extent did not vary significantly between cell batches. The overall responses resembled the pattern obtained for protein kinase B stimulation. MCP-1, MIP-1β, and IP10 induced a transient activation of ERK-2, whereas the response to ELC was more protracted. LARC, which did not affect protein kinase B, showed a weak activation of ERK-2. Similar results were obtained when the blots were developed with a specific antibody detecting diphosphorylated ERK-1 and ERK-2.


Signal transduction by CXC chemokine receptor 4. Stromal cell-derived factor 1 stimulates prolonged protein kinase B and extracellular signal-regulated kinase 2 activation in T lymphocytes.

Tilton B, Ho L, Oberlin E, Loetscher P, Baleux F, Clark-Lewis I, Thelen M - J. Exp. Med. (2000)

SDF-1 concentration dependence of prolonged protein kinase B and ERK-2 activation. (A) Western blot analysis of protein kinase B. T cells were stimulated with 10, 30, 100 nM or 1 μM SDF-1 for the indicated times. Activated (pSer473 PKB) and total protein kinase B (PKB) were determined as in the legend to Fig. 1 A. (B) Protein kinase B activity in immunoprecipitates from lysates of cells stimulated with the indicated concentrations of SDF-1 for either 1 min (•) or 20 min (○). Results are expressed as fold activation. (C) Western blot analysis of ERK-2. T cells were stimulated with 10, 100 nM and 1 μM SDF-1 for the indicated times. Activated ERK-2 (pERK-2) corresponds to the band with retarded electrophoretic mobility. Results are representative for at least three independent determinations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2193218&req=5

Figure 4: SDF-1 concentration dependence of prolonged protein kinase B and ERK-2 activation. (A) Western blot analysis of protein kinase B. T cells were stimulated with 10, 30, 100 nM or 1 μM SDF-1 for the indicated times. Activated (pSer473 PKB) and total protein kinase B (PKB) were determined as in the legend to Fig. 1 A. (B) Protein kinase B activity in immunoprecipitates from lysates of cells stimulated with the indicated concentrations of SDF-1 for either 1 min (•) or 20 min (○). Results are expressed as fold activation. (C) Western blot analysis of ERK-2. T cells were stimulated with 10, 100 nM and 1 μM SDF-1 for the indicated times. Activated ERK-2 (pERK-2) corresponds to the band with retarded electrophoretic mobility. Results are representative for at least three independent determinations.
Mentions: The prolonged activation of protein kinase B elicited by SDF-1 was unexpected and appeared to be a distinctive characteristic of the Gi protein–coupled receptor CXCR4. Therefore, we investigated whether SDF-1 causes protracted activation of other intracellular signal transduction pathways. Previous studies demonstrated that the MAP kinase kinase cascade leading to the activation of ERK-2 is regulated independently of protein kinase B 28, and chemokines were shown to stimulate ERK-2 activity in Jurkat cells 12. Using the same panel of chemokines, we compared the activation of ERK-2 in T cells. Fig. 1 D represents a Western blot analysis of ERK-2 activation performed with the same lysates as used in Fig. 1 A. Stimulation of ERK-2 results in the retarded electrophoretic mobility of the threonine- and tyrosine-phosphorylated enzyme that is detected by a specific antibody reacting with both resting and activated enzyme. Fig. 1 D shows that the most prominent and long-lasting effect was again obtained with SDF-1. With 100 nM SDF-1, phosphorylated ERK-2 was detectable for up to 90 min (see Fig. 4). The extent did not vary significantly between cell batches. The overall responses resembled the pattern obtained for protein kinase B stimulation. MCP-1, MIP-1β, and IP10 induced a transient activation of ERK-2, whereas the response to ELC was more protracted. LARC, which did not affect protein kinase B, showed a weak activation of ERK-2. Similar results were obtained when the blots were developed with a specific antibody detecting diphosphorylated ERK-1 and ERK-2.

Bottom Line: In addition, restimulation yields >60% of initial protein kinase B activity, indicating that the remaining receptors are not desensitized.Furthermore, activation is prolonged by inhibiting SDF-1 degradation.The sustained activation of cell survival and mitogenic pathways may account for the unique role of SDF-1 and CXCR4 in embryogenesis and lymphopoiesis.

View Article: PubMed Central - PubMed

Affiliation: Theodor Kocher-Institute, University of Bern, CH-3000 Bern 9, Switzerland.

ABSTRACT
We report that stromal cell-derived factor (SDF)-1 has the remarkable capacity to induce sustained signaling through CXC chemokine receptor 4 (CXCR4). In contrast to other chemokines, such as monocyte chemotactic protein 1 (CC chemokine receptor 2 [CCR2]), macrophage inflammatory protein 1beta (CCR5), liver and activation-regulated chemokine (LARC [CCR6]), Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC [CCR7]), and IP10 (CXCR3), SDF-1 stimulates the prolonged activation of protein kinase B and extracellular signal-regulated kinase (ERK)-2. Activation of protein kinase B is reversed by displacement of SDF-1 from CXCR4 or inhibition of phosphatidylinositol 3-kinase. Although increasing concentrations of SDF-1 enhance CXCR4 internalization, kinase activation is prolonged. In addition, restimulation yields >60% of initial protein kinase B activity, indicating that the remaining receptors are not desensitized. Furthermore, activation is prolonged by inhibiting SDF-1 degradation. The sustained activation of cell survival and mitogenic pathways may account for the unique role of SDF-1 and CXCR4 in embryogenesis and lymphopoiesis.

Show MeSH
Related in: MedlinePlus