Regulation of PKCK2 activity in TGF-β-mediated epithelial-mesenchymal transition

Abstract

E- to N-cadherin switching is a molecular hallmark of epithelial-mesenchymal transition (EMT) that might be a critical process in invasion and metastasis of cancer cells. Recently, it was reported that overexpression of PKCK2α catalytic subunit could induce E to N- cadherin switching. As it has been well known that TGF-β could induce the cadherin switching in A549, human non-small cell lung cancer cell line, this study was designed to examine whether PKCK2 is required for TGF-β mediated EMT. Results show that in the absence of PKCK2 activation, TGF-β could not induce the cadherin switching in A549 cells. PKCK2 activity began to increase after 24 hr of TGF-β treatment and achieved at its maximum at approximately 48 hr after treatment. TGF-β receptor I (TβRI) inhibitor abolished TGF-β mediated EMT and PKCK2 activation. As PKCK2 activity was increased, the expression of PKCK2 catalytic or regulatory subunit was examined. In the presence of TGF-β signaling, there was no change in the level of α catalytic subunit but the level of β regulatory subunit was down-regulated. β subunit knockout increases PKCK2 activity and induces E-to N-cadherin switching and imbalance between the expression level of catalytic subunit and regulator subunit could regulate PKCK2 activity. TGF-β signaling dependent down-regulation of CK2β was a proteasome dependent and TβRI kinase activity dependent process. C terminus of HSC70-Interacting Protein (CHIP) was found to be an E3 ubiquitin ligase of CK2β down regulation. As CK2β is phosphorylated by CK2α and phosphorylated CK2β was not down-regulated by TGF-β signaling. It was found that when CK2β is phosphorylated by CK2α, as yet unknown protein phosphatase could be involved for the dephosphorylation of CK2β and dephosphorylated CK2β could undergo CHIP mediated ubiquitination dependent degradation. The involvement of phosphatase was evidenced by the observation that protein phosphatase inhibitor, okadaic acid treatment could block CK2β down regulation induced by TGF-β signaling. Taken together, PKCK2 activation is required during TGF-β mediated EMT process. Imbalance between the expression level of CK2α and CK2β could regulate the PKCK2 activity. Therapeutic perturbation of PKCK2 activation in cancer cells could inhibit EMT and prevent metastasis.

E- to N- cadherin switching은 암세포의 전이와 침윤 과정에서 중요한 과정인 상피간엽전이 (EMT)의 분자적 특징이다. 최근에 PKCK2α catalytic subunit의 과 발현이 E- to N- cadherin switching을 유도함이 보고되었다. 비소세포형 폐암세포주인 A549에서 TGF-β가 cadherin switching을 유도한다는 것이 잘 알려져 있으므로, 본 연구는 TGF-β에 의한 상피간엽전이에서 PKCK2를 필요로 하는지에 대해 확인하고자 하였다. 먼저, A549 세포주에서 PKCK2 활성화가 없는 상황에서, TGF-β는 cadherin switching을 유발하지 못하였다. PKCK2 활성은 TGF-β 처리 후 24시간부터 증가하기 시작하여 처리 후 48시간에 최대로 증가하였다. 하지만 TGF-β receptor I (TβRI) 억제제는 TGF-β에 의한 상피간엽전이와 PKCK2 활성화를 억제하였다. 다음으로 PKCK2 활성이 증가될 때, PKCK2의 subunit의 발현을 확인하였다. TGF-β 신호전달이 있을 때, catalytic α sub unit의 발현수준에는 변화가 없지만 regulatory β subunit의 발현수준은 감소하였다. 또한 β subunit knockout은 PKCK2 활성을 증가시키고 E- to N- cadherin switching을 유도하였다. 이는 catalytic subunit과 regulatory subunit의 발현수준 사이의 불균형이 PKCK2 활성을 조절함을 보여주었다. 다음으로, TGF-β 신호전달에 의한 CK2β의 발현감소는 proteasome 의존적이고 TβRI kinase 활성에 의존적인 과정이라는 것을 확인하였다. 우선, C-terminus of HSC70-Interacting Protein (CHIP)이 CK2β의 발현감소에 작용하는 Ε3 ubiquitin ligase 라는 것을 발견하였다. CK2β는 CK2α에 의해 인산화 되는데, 인산화 된 CK2β는 TGF-β 신호전달에 의해 발현이 조절되지 않았다. 우리는 CK2β가 CK2α에 의해 인산화 될 때, 아직 밝혀지지 않은 protein phosphatase가 CK2β의 탈 인산화에 개입하며, 탈 인산화 된 CK2 β는 CHIP에 의해 유도되는 proteasome 의존적인 분해과정을 겪는다는 것을 확인하였다. 이러한 protein phosphatase의 개입은 protein phosphatase 억제제인 okadaic acid를 처리하였을 때 TGF-β 신호전달에 의해 유도되는 CK2β의 발현 감소가 억제된다는 것을 확인함으로 증명하였다. 종합하여 볼 때, PKCK2의 활성화는 TGF-β에 의한 상피간엽전이 과정 동안 필요하다. CK2α와 CK2β의 발현수준 사이의 불균형은 PKCK2 활성을 조절한다. 암세포에서 PKCK2 활성화의 치료적인 변화는 상피간엽전이를 억제하고 전이를 막는 것이다.