The Hippo pathway is evolutionarily conserved from Drosophila to mammals. FRMD6 is one crucial up- stream component of the Hippo pathway while its function and regulatory mechanism are largely elusive. We decided to purify the protein complex of FRMD6 to further explore its regulatory mechanism. We established the MCF7 breast cancer cells that stably expressed FRMD6 by retroviruses infection. And we purified the FRMD6-interacting protein complex for further analysis by high performance liquid chromatography-mass spectrometry/mass spectrometry（HPLC- MS/MS）. Interestingly, we observed that the major binding partner of FRMD6 is 14-3-3 family of proteins. The interac- tion between FRMD6 and 14-3-3 proteins was detected by co-immunoprecipitation（CO-IP）. The disruption of their interaction resulted in the nuclear localization of FRMD6. Importantly, the T28A mutant of FRMD6 showed stronger tumor suppressor function than wild type（WT） FRMD6. Our results indicate that 14-3-3 proteins tightly regulate the subcellular localization of FRMD6 so as to endow FRMD6 with the tumor suppressor function on breast cancer.
SAV1 is a core component involved in the Hippo pathway which can control the organ size via regulating cell proliferation and apoptosis simultaneously. We explored the regulatory mechanism of SAV1. We established the HEK293T cell pool, the cells in which can stably express SAV1 by retroviruses infection and found that SAV1 stable cells reduced the movement of themselves and resulted in multicellular aggregation. We purified SAV1 interacting protein complex using streptavidin resin and subsequently analyzed the digested peptides by high performance liquid chromatography（HPLC）-MS/MS. Results show that about 150 proteins were identified in the complex of SAV 1 with protein. TUBA1A, OTUD4, and ATD were identified as proteins interacting with SAV1. Importantly, PP1A, se- rine/threonine protein phosphatase PPl-alpha 1 catalytic subunit, was also in the top 10 list. The interaction between PP1A and SAV1 was detected by both co-immunoprecipitation（CO-IP） and immunostaining. Our results indicate that PP1A might be the phosphatase of SAV1 and may take part in the regulation of the Hippo pathway.
In order to further investigate the role of ClC-2（ClC=chloride-ion channel） played in the regulation of cell proliferation and differentiation, the capablity of ClC-2 phosphorylation catalyzed by mitogen-activated protein ki-nase（MAPK） was studied. A mutation of 659Ser to Ala（S659A） of the rabbit ClC-2 cDNA in the consensus sequence of MAPK phosphorylation was introduced by overlap extension polymerase chain reaction（PCR）. Recombinant vectors pGEX-4T-1/ClC-2-2CT and pGEX-4T-1/ClC-2CT（S659A） were constructed. They were transformed to E. coli BL21, expressed by isopropy-β-D-thiogalactoside（IPTG） induction, the recombinant proteins were subjected to purification by glutathione sepharose 4B affinity chromatography. In vitro phosphorylation of the fusion proteins catalyzed by MAPK was performed. The results show that fusion protein GST/ClC-2CT（wild type） can be phosphorylated by MAPK, and this phosphorylation can be restrained by the inhibitor p42/44MAPK, PD98095; while the phosphorylation level of fusion protein GST/ClC-2CT（S659A）（mutant） was significantly reduced. Therefore, ClC-2 can be phosphorylated by MAPK and the target site of the phosphorylation is most likely the 659Ser residue.
