Cbl and Akt regulate CXCL8-induced and CXCR1- and CXCR2-mediated chemotaxis.
CXCL8 (IL-8) plays an important role in the pathogenesis of a variety of inflammatory diseases. However, little is known about the signaling pathways that regulate CXCL8-induced chemotaxis. Here, we found that CXCL8 treatment of CXCR1- and CXCR2-over-expressing L1.2 cells (CXCR1-L1.2 and CXCR2-L1.2, respectively) induced the phosphorylation of Cbl and Akt. The tyrosine kinase inhibitor Tyrphostin A9, phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 as well as proteasome inhibitors significantly blocked the CXCL8-induced chemotaxis of L1.2 cells and human neutrophils. We further found that stimulation with CXCL8 enhanced the association of the PI3K subunit p85 with Cbl. Additionally, over-expression of wild-type Cbl and G306E-Cbl (mutation in the tyrosine kinase-binding domain) inhibited chemotaxis by approximately 50% as compared with the vector control, whereas the 70Z mutant (deletion in the RING finger domain) did not reduce migration. However, wild-type Cbl or its mutants had no effect on the CXCL8-induced activation of MAPK, indicating that Cbl specifically modulated CXCL8-induced chemotaxis. Furthermore, over-expression of the kinase-dead Akt mutant decreased CXCL8-induced chemotaxis by 60% and diminished Cbl phosphorylation as compared with the vector control. The CXCL8-induced phosphorylation of Cbl was also reduced when cells were pre-treated with the PI3K inhibitor LY294002. Lastly, we have shown that pre-treatment of L1.2 cells with the proteasome inhibitor Lactacystin blocks CXCL8-induced internalization of the CXCR1 and CXCR2 receptors. These studies provide new information regarding CXCL8-induced signaling pathways that may regulate chemotaxis and receptor internalization.
Lane
Heather C
HC
Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Room 343, Harvard Medical School, 4 Blackfan Circle, Boston, MA 02115, USA.
Anand
Appakkudal R
AR
Ganju
Ramesh K
RK
D000818:Animals; D002633:Chemotaxis / Q000276:immunology / Q000502:physiology*; D016209:Interleukin-8 / Q000276:immunology*; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl / Q000276:immunology / Q000378:metabolism*; D023062:Receptors, Interleukin-8A / Q000276:immunology*; D023063:Receptors, Interleukin-8B / Q000276:immunology*; D054462:p21-Activated Kinases
D016209:Interleukin-8; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D019869:Phosphatidylinositol 3-Kinases; D010749:Phosphoprotein Phosphatases; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D051057:Proto-Oncogene Proteins c-akt; D050721:Proto-Oncogene Proteins c-cbl; D023062:Receptors, Interleukin-8A; D023063:Receptors, Interleukin-8B; D054462:p21-Activated Kinases; C515907:PAK2 protein, human; C515909:Pak2 protein, mouse; C493849:Cbl protein, mouse