Proapoptotic function of the nuclear Crk II adaptor protein.
Crk II and Crk L have both cytosolic and nuclear functions. While Crk L is a bona fide nuclear signaling protein because of its ability to bind tyrosine-phosphorylated STAT5 and act as a transcriptional coactivator, the function of nuclear Crk II is less well understood. The present study was undertaken to investigate whether Crk II is in the nucleus, how Crk II translocates into the nucleus, whether it possesses a functional NES, and to determine if nuclear Crk II affects cell cycle checkpoints and promotes apoptosis. Toward this goal, we used several independent techniques to show that a significant percentage of the total endogenous Crk II partitions in the nucleus in mammalian cells, where it forms distinct complexes with DOCK180, Wee1, and Abl. We found no evidence that Crk II bound to Crm1 nor that the localization of GFP-Crk II was sensitive to LMB, an inhibitor of Crm1. To better define the significance of nuclear Crk II localization, we generated a GFP-Crk II protein (GFP-Crk-nuc) fused to three tandem nuclear localization signals derived from the SV40 large T-antigen. GFP-Crk-nuc exhibited exclusive nuclear localization, and in contrast to wild-type Crk, GFP-Crk-nuc expressing cells could not be propagated upon selection in G418-containing media, suggesting nuclear accumulation of Crk II caused either growth arrest or apoptosis. When transiently transfected cells were FACS sorted, GFP-expressing cells showed defective cell adhesion on tissue culture surfaces and showed an increased level of apoptosis assessed by pycnotic nuclei, annexin V staining, and PARP cleavage. Although we found that Crk II bound to the cell cycle protein Wee1, expression of GFP-Crk-nuc did not induce a G2/M cell cycle block or cause increased Cdc2 Tyr15 phosphorylation. Finally, upon UV stimulation, we found that endogenous Crk II translocated to the nucleus and potentiated the extent of UV-inducible apoptosis after 4 h. These data suggest that nuclear compartmentalization of Crk II antagonizes its cytoskeletal functions and assign a proapoptotic role to the nuclear pool of Crk II.
Kar
Bishnupriya
B
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey 07103, USA.
Reichman
Charles T
CT
Singh
Sukhwinder
S
O'Connor
J Patrick
JP
Birge
Raymond B
RB
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D017209:Apoptosis / Q000235:genetics* / Q000528:radiation effects; D002448:Cell Adhesion / Q000235:genetics; D002451:Cell Compartmentation; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D021382:Protein Sorting Signals / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D050737:Proto-Oncogene Proteins c-crk / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D014157:Transcription Factors; D014466:Ultraviolet Rays; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D000097606:Exportin 1 Protein
D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D021382:Protein Sorting Signals; D011505:Protein-Tyrosine Kinases; D016315:Proto-Oncogene Proteins c-abl; D050737:Proto-Oncogene Proteins c-crk; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D020744:rac GTP-Binding Proteins; D000097606:Exportin 1 Protein; C082869:CRKL protein; C099818:DOCK1 protein, human; C499349:WEE1 protein, human