Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes.
Haematopoietic cell kinase (Hck) is a protein tyrosine kinase of the Src family specifically expressed in phagocytes as two isoforms, p59Hck and p61Hck, present at the plasma membrane and lysosomes, respectively. We report that ectopic expression of a constitutively active mutant of p61Hck (p61Hck(ca)) triggered the de novo formation of actin-rich rings at the ventral face of the cells that we characterized as bona fide podosome rosettes, structures involved in cell migration. Their formation required the adaptor domains and the kinase activity of p61Hck, the integrity of microfilament and microtubule networks and concerted action of Cdc42, Rac and Rho. Podosome rosette formation was either abolished when p61Hck(ca) was readdressed from lysosomes to the cytosol or triggered when p59Hck(ca) was relocalized to lysosomes. Lysosomal markers were present at podosome rosettes. By stimulating exocytosis of p61Hck(ca) lysosomes with a calcium ionophore, the formation of podosome rosettes was enhanced. Interestingly, we confirm that, in human macrophages, Hck and lysosomal markers were present at podosomes which were spatially reorganized as clusters, a foregoing step to form rosettes, upon expression of p61Hck(ca). We propose that lysosomes, under the control of p61Hck, are involved in the biogenesis of podosomes, a key phenomenon in the migration of phagocytes.
Institut de Pharmacologie et de Biologie Structurale - Centre National de la Recherche Scientifique UMR 5089, Département Mécanismes Moléculaires des Infections Mycobactériennes, 205 route de Narbonne, 31077 Toulouse cedex, France.
Céline
C
Cougoule
Sébastien
S
Carréno
Jerôme
J
Castandet
Arnaud
A
Labrousse
Catherine
C
Astarie-Dequeker
Renaud
R
Poincloux
Véronique
V
Le Cabec
Isabelle
I
Maridonneau-Parini
D000199:Actins / Q000032:analysis; D026905:Amino Acid Transport Systems / Q000032:analysis; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002465:Cell Movement / Q000502:physiology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000502:physiology; D020536:Enzyme Activators / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D051379:Mice; D041681:NIH 3T3 Cells; D015739:Nocodazole / Q000494:pharmacology; D019000:Phosphotyrosine / Q000032:analysis; D010957:Plasmids; D020033:Protein Isoforms / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D027981:Symporters / Q000032:analysis; D016596:Vinculin / Q000032:analysis; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D026905:Amino Acid Transport Systems; D020536:Enzyme Activators; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; C483904:Slc36a1 protein, mouse; D027981:Symporters; D016596:Vinculin; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D015638:Cytochalasin D; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; C496632:Hck protein, mouse; D051572:Proto-Oncogene Proteins c-hck; D001619:beta-N-Acetylhexosaminidases; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; D015739:Nocodazole