KIBRA Protein Phosphorylation Is Regulated by Mitotic Kinase Aurora and Protein Phosphatase 1

Abstract

Recent genetic studies in Drosophila identified Kibra as a novel regulator of the Hippo pathway, which controls tissue growth and tumorigenesis by inhibiting cell proliferation and promoting apoptosis. The cellular function and regulation of human KIBRA remain largely unclear. Here, we show that KIBRA is a phosphoprotein and that phosphorylation of KIBRA is regulated in a cell cycle-dependent manner with the highest level of phosphorylated KIBRA detected in mitosis. We further demonstrate that the mitotic kinases Aurora-A and -B phosphorylate KIBRA both in vitro and in vivo. We identified the highly conserved Ser 539 as the primary phosphorylation site for Aurora kinases. Moreover, we found that wild-type, but not catalytically inactive, protein phosphatase 1 (PP1) associates with KIBRA. PP1 dephosphorylated Aurora-phosphorylated KIBRA. KIBRA depletion impaired the interaction between Aurora-A and PP1. We also show that KIBRA associates with neurofibromatosis type 2/Merlin in a Ser 539 phosphorylation-dependent manner. Phosphorylation of KIBRA on Ser 539 plays a role in mitotic progression. Our results suggest that KIBRA is a physiological substrate of Aurora kinases and reveal a new avenue between KIBRA/Hippo signaling and the mitotic machinery.The Hippo signaling pathway, originally defined in Drosophila, controls organ size, tumorigenesis, and cell contact inhibition by regulating cell proliferation and apoptosis (1-3). In mammalian cells, kinases Mst1/2 (orthologs of Drosophila Hippo) phosphorylate and activate Lats1/2 (orthologs of Warts) (4, 5). Lats, in turn, phosphorylates and inactivates the downstream effectors YAP/TAZ (orthologs of Yorkie) (6 -9). The transcriptional coactivators YAP and TAZ function together with transcription factors such as TEAD1-4 (Scalloped in Drosophila) (10 -13) to induce target gene expression, including Birc5 (8), cytokines such as connective tissue growth factor (10,14), and the EGF family member amphiregulin (15). Accumulated evidence suggests that this emerging signaling pathway plays a critical role in cancer development with the most evident contribution to hepatocellular carcinoma (1, 2, 16). For example, mice lacking Lats1 or WW45 (ortholog of Salvador) develop several types of tumors (17)(18)(19). Overexpression of YAP or loss of Mst1 and Mst2 in mouse liver dramatically increases the organ size and eventually induces hepatocellular carcinoma (8, 20 -23). The YAP locus is consistently amplified, and elevated YAP expression has been observed in many human cancers, including liver cancers (8, 14, 24 -26).KIBRA, a WW domain-containing protein (27), was originally identified as a memory performance-associated protein (28 -30). The physiological function of human KIBRA is not well understood, although it has been shown to play a role in podocyte migration (31, 32) and to be involved in age-dependent risk of Alzheimer disease (33). It was also reported to interact with discoidin domain receptor 1 to modulate collageninduced signaling (34). Interestingly, KIBRA expression wa...

KIBRA Regulates Aurora Kinase Activity and Is Required for Precise Chromosome Alignment During Mitosis

“…Plasmids-The human KIBRA, Mst1, Lats1, Lats2, Aurora-A, and Aurora-B constructs and their corresponding derivatives have been described previously (27,40). Truncated constructs were made by PCR and verified by sequencing and restriction enzyme digestion.…”

“…Furthermore, we showed that KIBRA is phosphorylated by Aurora-A and -B kinases during mitosis (40). Functions of Aurora kinases and Lats2 in mitosis are well defined, but whether KIBRA has a mitotic role is currently unknown.…”

Oncoprotein YAP Regulates the Spindle Checkpoint Activation in a Mitotic Phosphorylation-dependent Manner through Up-regulation of BubR1

“…In Vitro Dephosphorylation/Phosphatase Assay-GST-YAP was first phosphorylated by CDK1/cyclin B in vitro as described (30). Phosphorylated GST-YAP was pulled down by glutathioneagarose (Santa Cruz Biotechnology, Dallas, TX), and the dephosphorylation assay was performed as previously described except 32 P was replaced by the phospho-antibodies (30).…”

“…Phosphorylated GST-YAP was pulled down by glutathioneagarose (Santa Cruz Biotechnology, Dallas, TX), and the dephosphorylation assay was performed as previously described except 32 P was replaced by the phospho-antibodies (30). Antibodies, Immunoprecipitation, and Western Blot AnalysisThe YAP antibodies from Abnova (Taipei, Taiwan; catalog no.…”

“…Anti-Thr 288 Aurora-A/Thr 232 Aurora-B, antiSer 127 YAP, and anti-Ser 10 H3 were from Cell Signaling Technology (Danvers, MA). Immunoprecipitation and Western blotting assays were done as described (30).…”