Proteomic analysis of annexin A2 phosphorylation induced by microtubule interfering agents and kinesin spindle protein inhibitors.
Microtubule interfering agents (MIAs) are antitumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause the accumulation of mitotic cells and subsequently cell death. We used two-dimensional gel electrophoresis (2DE) followed by MALDI-MS analysis to demonstrate that the MIAs vinblastine (Velban) and paclitaxel (Taxol), as well as the KSP inhibitor S-tritil-L-cysteine, induce the phosphorylation of annexin A2 in human lung carcinoma A549 cells. Further tandem mass spectrometry analysis using a combination of peptide fragmentation methods (CID and ETD) and multiple reaction monitoring (MRM) analysis determined that this modification occurs mainly at threonine 19. We show that MIAs and KSP inhibitors only induce this phosphorylation in cells capable of reaching the M phase. Furthermore, we demonstrate that CDK activity is required for the phosphorylation of annexin A2 induced by MIAs and KSP inhibitors. Finally, we have used double thymidine block synchronization to demonstrate that annexin A2 is not phosphorylated during a normal mitosis, indicating that this phosphorylation of annexin A2 is a specific response to these drugs.
Fernandez-Garcia
Belen
B
Departamento de Bioquímica y Biología Molecular and Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Universidad de Oviedo, 33071 Oviedo, Spain.
Casado
Pedro
P
Prado
Miguel A
MA
Ugarte-Gil
Lorea J
LJ
Artime
Noelia
N
Cabal-Hierro
Lucía
L
Calvo
Enrique
E
López
Juan Antonio
JA
Ramos
Sofía
S
Lazo
Pedro S
PS
D000595:Amino Acid Sequence; D017306:Annexin A2 / Q000737:chemistry / Q000378:metabolism*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D003545:Cysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects*; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000494:pharmacology; D010449:Peptide Mapping; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D050257:Tubulin Modulators / Q000494:pharmacology*; D014747:Vinblastine / Q000494:pharmacology
C472822:ANXA2 protein, human; D017306:Annexin A2; C470243:KIF11 protein, human; D050257:Tubulin Modulators; D010769:Phosphothreonine; C003724:3-tritylthio-L-alanine; D014747:Vinblastine; D018844:Cyclin-Dependent Kinases; D016547:Kinesins; D003545:Cysteine; D017239:Paclitaxel