c-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration

Abstract

Cytoskeleton dynamics are regulated by Src-family tyrosine kinases (SFKs) and c-Abl. We found that the SFK members Hck and c-Fgr regulate tyrosine phosphorylation of c-Abl and c-Abl associates with beta1 integrin-bound Hck or c-Fgr in murine macrophages. Studies with selective inhibitors and cells from SFK-deficient mice showed that c-Abl and SFK regulate migration and activation of the small GTPases Cdc42 and Rac in macrophages. Additionally, human neutrophil chemotactic activity was reduced by c-Abl inhibitors, and neutrophils from chronic myeloid leukaemia patients displayed an increased chemotactic ability. Hence, Src-family kinase and c-Abl cross-talk in the regulation of myeloid cell migration.

Fig. 1

c-Abl is a substrate of SFKs in BMDM and binds to c-Fgr or Hck complexed to the β1 integrin chain. (A) BMDM, isolated and cultivated as described in Section 2, were lysed and anti-c-Abl immunoprecipitates (IP) separated on SDS/PAGE gels. Proteins were blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). One representative experiment of 2–4 performed is reported. (B and C) BMDM lysates were incubated with anti-β1 integrin Ab and immunoprecipitates (IP) processed as described above with Abs of the indicated specificity (WB). WT, wild-type BMDM; HF^−/−, hck−/−fgr−/− BMDM. Two representative experiment of three performed are reported.

Fig. 2

Macrophage migration requires the c-Abl kinase activity. (A–D) BMDM monolayers were wounded with the tip of a pipette and, after washing, added with medium supplemented with 100 ng/ml LPA without (A, B) or plus 10 μM imatinib mesylate (STI) (C) or 10 μM PP2 (D). Monolayers were photographed immediately after the wound (A) or after 6 h from wounding (B–D). (E) Cells migrated into the wound were quantified after different time from wounding. (F) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. To detect phosphorylation of SFKs, lysates were separated on SDS/PAGE gels, proteins blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). To detect c-Abl tyrosine phosphorylation, BMDM were lysed in RIPA buffer as described in Section 2 and immunoprecipitated (IP) with anti-Abl Abs before blotting. (G) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. (H) Phase contrast images of cells at the margin of the wound after 2 h are shown. These were taken at 40× magnification with an Olympus IX50 microscope and acquired with an Olympus c-7070 wide zoom Digital compact camera. Inhibitors were used at the concentration reported above. One typical of several experiment performed is reported. (I) Baf3 cells expressing Bcr/Abl are strongly polarized. Baf3 cells transfected with an empty vector (Baf3/pSrl) or a vector containing wild-type Bcr/Abl (Baf3/p210wt) or Bcr-Abl with the T315 mutation (Baf3/T315) were cultivated and incubated with STI (10 μM) or PP2 (10 μM) as described in Section 2. Morphology of cells either untreated or treated with STI or PP2 is shown. Cells were plated in 24 well plates in RPMI 1640 medium containing 10% FBS and phase contrast images at 40× magnification taken as described in the legend.

Fig. 3

SFKs an c-Abl regulate Cdc42 and Rac activation. BMDM were isolated, cultivated and lysed as described in Section 2. GTP-bound Cdc42 and Rac were assayed after 30 min of incubation with 100 ng/ml LPA without or with 10 μM imatinib mesylate as described in Section 2 and mean result of three independent experiments are shown.

Fig. 4

c-Abl regulates human neutrophil migration and polarization. (A) Neutrophils were incubated with 1 μM imatinib (STI) or bosutinib (SKI) for 30 min before transfer to the upper compartment of 3 μm pore transwells. Neutrophils migrated to the lower compartment of the transwell that contained or not (Ctr) 100 nM fMLP were quantified after 60 min. Mean results ± S.D. of three independent experiments are reported. (B) Control or imatinib (STI)-treated neutrophils were stimulated with 100 nM fMLP for 5 min. (C) Neutrophils from healthy donors or first diagnosis CML patients were assayed as in (A).