Analysis of the subcellular distribution of avian p95-APP2, an ARF-GAP orthologous to mammalian paxillin kinase linker.
We describe here the identification and characterization of avian p95-APP2, a multi-domain protein of a recently identified family of ADP-ribosylation factor (ARF)-GTPase-activating proteins (GAPs) including mammalian G protein-coupled receptor kinases (GRK)-interactor 1 (GIT1), paxillin kinase linker (PKL), and GIT2, as well as avian p95-APP1. The p95-APP2 is eluted from Rac-GTP-gamma-S, but not from Rac-GDP-beta-S columns. As other members of the family, p95-APP2 has binding regions for the focal adhesion protein paxillin, and for the Rac exchanging factor PIX. Sequence comparison indicates that p95-APP2 is the avian orthologue of mammalian PKL. Expression studies showed a largely diffuse distribution of the full length p95-APP2, without evident effects on cell morphology. We observed a dramatic difference between the localization of the amino-terminal portion of the protein, including the ARF-GAP domain and the three ankyrin repeats, and the carboxy-terminal portion including the paxillin-binding site. Moreover, the expression of truncated carboxy-terminal polypeptides including both the PIX- and paxillin-binding regions leads to a marked localization of the protein together with paxillin at large vesicles. Comparison of the expression of corresponding ARF-GAP-deficient constructs from p95-APP2 and p95-APP1 shows their distribution at distinct endocytic compartments. Altogether, these data support a role of distinct members of this family of ARF-GAPs in the regulation of different steps of membrane traffic during cell motility, and suggest that p95-APP2 may shuttle between an intracellular compartment and the cell periphery, although, further work will be needed to address this point.
Paris
Simona
S
Cell Adhesion Unit, DIBIT, S. Raffaele Scientific Institute, Via Olgettina 58, 20132 Milan, Italy.
Za
Lorena
L
Sporchia
Barbara
B
de Curtis
Ivan
I
D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010750:Phosphoproteins; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013045:Species Specificity; D013347:Subcellular Fractions / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C485647:GIT1 protein, human; D020690:GTPase-Activating Proteins; D046911:Macromolecular Substances; D010750:Phosphoproteins; D020727:ADP-Ribosylation Factors