Expression And Function Of Integrin-linked Kinase-associated Phosphatase (ILKAP)

Integrin-linked kinase-associated phosphatase is a novel member of protein serine/threonine phosphatases 2C family. ILKAP negatively regulates the integrin linked kinase 1 signaling pathway through inhibiting the activity of integrin linked kinase 1, so that the tumor cells accumulate in G1 phase, which inhibits their proliferation and growth as non-anchor and changes the characteristics of tumor cells. On the other hand, ILKAP regulates apoptosis signal JNK /SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) pathway through the activation Thr845 of ASK1 (apoptosis signal-regulating kinase 1,ASK1)to promote apoptosis of tumor cells. There is another significant protein named palladin that plays an important role in the tumor occurrence and development. Numerous recent studies have suggested that palladin is involved in the invasive cell motility such as metastatic cancer cells.In this thesis, according to the result that ILKAP molecule is associated with palladin in yeast two-hybrid screening, ILKAP was first expressed in Escherichia coli BL21 (DE3) and purified to over 95% purity. Western blot analysis verified expressed ILKAP and the specific activity of purified ILKAP was 34.5U/mg.The antiserum against ILKAP was then prepared due to the lack of commercial anti-ILKAP specific antibody. Two rabbits were immunized six times by intramuscular and intravenous injection and the prepared serum antibody titer was analyzed by Western blot and indirect enzyme-linked immunosorbent assay method (indirect ELISA). The results showed that the effective anti-ILKAP antiserum was obtained and the serum antibody titer reached 1:1600.In order to investigate the interaction between ILKAP and palladin, the eukaryotic expression vector of ILKAP, pcDNA3.1 (+)– His-ILKAP was also constructed. Then the intracellular location of ILKAP was observed using cos7 cells. Meanwhile, the localization of palladin in cos7 cells was also analyzed. The immunofluorescence assay appeared that ILKAP in cos7 cells located in the nucleus region while palladin did in the cytoplasm region. After determining the localization of ILKAP and palladin in target cells, we carried out cotransfection with pcDNA3.1 (+)-His-ILKAP and pcDNA3.1(+)-flag-palladin. The immunoprecipitation experiments were carried out to verify the interaction between ILKAP and palladin in eukaryotic cells. The result showed that there was the stronge interaction between ILKAP and paladin in eukaryotic cells.So far, no one study has been reported on the interaction of these two proteins. However, many results have proved that both palladin and ILKAP have relation to the migration and movement of tumor cells. With the further study of ILKAP function, more and more attention will be paid to the occurrence and development of tumor. In the future, we may inhibit cancer cell metastasis by regulating the function of ILKAP or palladin.The study on tumor occurrence and development are hotspots in recent years. Oncogenes and tumor suppressor genes play an important role in tumor occurrence, development and treatment. With the further study of ILKAP, the role of ILKAP in tumor occurrence and development would attract scientists'attention.