Identification of V23RalA-Ser194 as a critical mediator for Aurora-A-induced cellular motility and transformation by small pool expression screening.
Human Aurora kinases have three gene family members: Aurora-A, Aurora-B, and Aurora-C. It is not yet established what the specificity of these kinases are and what signals relayed by their reactions. Therefore, we employed small pool expression screening to search for downstream substrates of Aurora-A. Interestingly, all of the identified Aurora-A substrates were resistant to serve as substrates for Aurora-B or Aurora-C, suggesting that these Aurora family members may have distinct substrate specificity for propagation of diverse signaling pathways, even though they share a conserved catalytic kinase domain. Of the candidate substrates, Aurora-A could increase the functional activity of RalA. Mutational analysis revealed that RalA-Ser194 was the phosphorylation site for Aurora-A. Ectopic expression of V23RalA-WT could enhance collagen I-induced cell migration and anchorage-independent growth in Madin-Darby canine kidney (MDCK) Aurora-A stable cell lines. In contrast, overexpression of V23RalA-S194A in MDCK Aurora-A-stable cell lines abolished the intrinsic migration and transformation abilities of Aurora-A. To our knowledge, this is the first systematic search for the downstream substrates of Aurora-A kinase. Moreover, these results support the notion that Aurora-A may act in concert with V23RalA through protein phosphorylation on Ser194 to promote collagen I-induced cell motility and anchorage-independent growth in MDCK epithelial cells.
Wu
Jiunn-Chyi
JC
Division of Molecular and Genomic Medicine, National Health Research Institutes, 35, Keyan Road, Zhunan Town, Miaoli County 350, Taipei, ROC.
Chen
Tzong-Yueh
TY
Yu
Chang-Tze R
CT
Tsai
Si-Jie
SJ
Hsu
Jung-Mao
JM
Tang
Ming-Jer
MJ
Chou
Chen-Kung
CK
Lin
Wey-Jinq
WJ
Yuan
Chiun-Jye
CJ
Huang
Chi-Ying F
CY
D000595:Amino Acid Sequence; D000818:Animals; D064096:Aurora Kinase A; D064107:Aurora Kinase B; D064108:Aurora Kinase C; D064127:Aurora Kinases; D018797:Cell Cycle Proteins; D002460:Cell Line; D002465:Cell Movement; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007668:Kidney; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection; D029867:Xenopus Proteins; D020725:ral GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D011494:Protein Kinases; C112134:AURKA protein, Xenopus; C576921:AURKB protein, human; C576927:AURKC protein, human; C576918:Aurka protein, mouse; C576920:Aurka protein, rat; C576924:Aurkb protein, mouse; C576925:Aurkb protein, rat; C576928:Aurkc protein, mouse; D064096:Aurora Kinase A; D064107:Aurora Kinase B; D064108:Aurora Kinase C; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases; C492198:RALA protein, human; D020725:ral GTP-Binding Proteins