Tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) by Hck regulates macrophage function.
We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck(-/-) bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck(-/-) BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP(-/-) BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.
From the Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461 and.
Haein
H
Park
Athanassios
A
Dovas
Samer
S
Hanna
Claire
C
Lastrucci
Celine
C
Cougoule
Romain
R
Guiet
Isabelle
I
Maridonneau-Parini
Dianne
D
Cox
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology; D002460:Cell Line; D002465:Cell Movement; D002633:Chemotaxis; D002634:Chemotaxis, Leukocyte; D003094:Collagen / Q000737:chemistry; D003433:Crosses, Genetic; D042783:Endothelial Cells / Q000166:cytology; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism; D008648:Mesoderm / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry; D051572:Proto-Oncogene Proteins c-hck / Q000378:metabolism*; D034622:RNA Interference; D057705:Transendothelial and Transepithelial Migration; D014443:Tyrosine / Q000737:chemistry; D051316:Wiskott-Aldrich Syndrome Protein / Q000737:chemistry* / Q000378:metabolism*
D020033:Protein Isoforms; C495768:Was protein, mouse; D051316:Wiskott-Aldrich Syndrome Protein; D014443:Tyrosine; D003094:Collagen; C496632:Hck protein, mouse; D051572:Proto-Oncogene Proteins c-hck
Chemotaxis; Extracellular Matrix; Hck; Invasion; Macrophage; Nonreceptor Tyrosine Kinase; Phagocytosis; WASP