Functional Interaction Between C-Abl And Plk1

Mammalian non-receptor tyrosine kinase c-Abl is a cellular homolog of the v-abloncogene of Abelson murine leukemia virus. c-Abl has been implicated in theregulation of the cell cycle, apoptosis, adhesion, migration, cytoskeletalreorganization and DNA damage responses. Plk1is a serine/threonine kinase that iswidely expressed in eukaryotic cells and regulates mitosis, cytokinesis, DNA damageresponses and tumorigenesis through its phosphorylation of many substrates. c-Abland Plk1both play important roles in cell signal transduction and can regulate thecell cycle and apoptosis via their kinase activity. The fact that both c-Abl and Plk1are regulated by the ATM signaling pathway in the DNA damage response and canregulate Rad51during homologous genetic recombination promoted us to investigatewhether there is a relationship between c-Abl and Plk1. Our previous data showedthat the tubulin level decreased markedly when c-Abl and its homologous gene Argwere knocked down. Indeed, the assembly of a normal microtubule structure andspindle assembly were defective, leading to many polykaryocytes and influencingcytokinesis. Because c-Abl and Plk1interact when overexpressed, we investigatedthe molecular mechanism of c-Abl and Plk1interaction and its biological effect toprovide evidence for the roles these kinases in cell cycle regulation.We observed that endogenous c-Abl specifically co-immunoprecipitated with Plk1,and this co-IP was reciprocal. Using PLA method, we further found that c-Abl caninteract with Plk1under physiological conditions. We found that c-Abl and Plk1interacted with each other when they were overexpressed and that their interactionmainly depends on c-Abl kinase activity and partially depends on Plk1kinaseactivity. A direct interaction between c-Abl and Plk1was demonstrated by GSTpull-down and far-western assays, showing that c-Abl interacted with Plk1throughits SH2and SH3domains and that Plk1interacted with c-Abl through its PBDdomain. Using point mutation and co-IP, GST-pull down,we showed that Plk1interacted with c-Abl mainly via Plk1H538and K540. Plk1was phosphorylated byc-Abl when c-Abl and Plk1were overexpressed, and this phosphorylation mainly depended on c-Abl kinase activity and partly depended on Plk1kinase activity. c-Ablcan phosphorylate the KD domain of Plk1. LC-MS/MS indicated that c-Abl canphosphorylate Plk1at residues Y203, Y217, Y268and Y570.We found that the level of Plk1was decreased when c-Abl and Arg were knockeddown. However, if c-Abl was recovered in DKO cells, the Plk1level alsosubsequently recovered, and c-Abl could improve the level of Plk1when they wereoverexpressed. Using quantitative PCR and CHX assays, we found that c-Ablpromoted the level of Plk1post-transcriptionally and not transcriptionally and thatc-Abl can inhibit the degradation of Plk1through the ubiquitin-proteasome pathway.As Plk1kinase activity is tightly linked to its expression level, we addressedwhether c-Abl could regulate Plk1activity. The results of co-IP and IB revealed thatthe level of p-Plk1T210was decreased when the kinase activity of c-Abl wasinhibited either endogenously or exogenously. Through cell synchronization, wedemonstrated that c-Abl can regulate the level of p-Plk1T210, which would furtherinfluence Plk1phosphorylation of such substrates as TCTP and CDC25C.It was shown by an immunofluorescence assay that c-Abl does not regulate thecellular location of Plk1during prophase, prometaphase, metaphase, anaphase ortelophase. Cell synchronization preliminary showed that c-Abl may regulate the levelof Plk1, which would influence cell entry into mitosis.We found that c-Abl kinaseactivity can regulate mitotic progression by time lapse technique.The interaction between c-Abl and Plk1provides a feedback mechanism, thusPlk1can also regulate the level of c-Abl. Within a certain range, the level ofendogenous c-Abl increased in a dose-dependent manner when Plk1wasoverexpressed, whereas Plk1inhibited the level of c-Abl when Plk1expressionexceeded this range. Quantitative PCR showed that Plk1enhanced c-Abl at thetranscriptional level.In summary, we showed that c-Abl can interact with Plk1and can specificallyphosphorylate Plk1, thus regulating the level and activity of this kinase. c-Abl canregulate the level of Plk1to influence the G2/M conversion, and its kinase activitycan regulate mitotic progression. These findings provide new insight into the roles ofthe c-Abl and Plk1kinases and the mechanism of G2/M and mitosis regulation.