Re-evaluating the role of Tao1 in the spindle checkpoint.
The spindle checkpoint restrains anaphase onset and mitotic exit until all chromosomes are stably attached to the mitotic spindle via their kinetochores. The Tao1 protein kinase was recently reported as a novel spindle checkpoint component. When an siRNA was used to repress Tao1, the essential spindle checkpoint component Mad2 failed to localise to kinetochores, and cells rapidly exited mitosis. Tao1 was also shown to interact with BubR1, another essential checkpoint component, and be rapidly degraded after mitosis, a feature typical of many mitotic regulators. Here, we identify four different siRNAs that repress Tao1 protein levels as efficiently as the previously reported siRNA. However, these siRNAs do not override the spindle checkpoint. We also present data indicating that Tao1 does not interact with BubR1 and that it is not rapidly degraded after mitosis. We show that the previously reported siRNA not only represses Tao1 but also dramatically reduces Mad2 protein levels. Crucially, expression of exogenous Mad2, but not Tao1, rescued the spindle checkpoint phenotype induced by this siRNA. Thus, the key functional data implicating Tao1 in the spindle checkpoint can be explained by an off-target siRNA phenomenon that results in Mad2 inhibition. Taken together, our data do not support the notion that Tao1 is a component of the spindle checkpoint.
Westhorpe
Frederick G
FG
University of Manchester, UK.
Diez
Maria A
MA
Gurden
Mark D J
MD
Tighe
Anthony
A
Taylor
Stephen S
SS
D000705:Anaphase; D002135:Calcium-Binding Proteins / Q000378:metabolism; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002877:Chromosomes, Human / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism*; D064246:Mad2 Proteins; D008938:Mitosis; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C577488:CDC20 protein, human; D011494:Protein Kinases; C584277:BUB1 protein, human; C090464:Bub1 spindle checkpoint protein; D017346:Protein Serine-Threonine Kinases; C115483:TAO1 protein kinase; D020930:MAP Kinase Kinase Kinases