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PLC-r의 활성인자인 Ahnak단백질에 대한 특성 연구

Title
PLC-r의 활성인자인 Ahnak단백질에 대한 특성 연구
Authors
홍지영
Issue Date
2000
Department/Major
대학원 분자생명과학부
Publisher
이화여자대학교 대학원
Degree
Master
Abstract
다양한 기능을 수행하는 세포는 growth factor나 hormone의 자극에 대하여 단순한 신호전달체계를 갖지 않고 세포신호전달체계사이의 의견교환 즉 "cross-talk"을 수행함으로써 세포의 신호전달 자체뿐만 아니라 신호전달의 강도 (strength)를 조절한다. 최근 neurological tissue에서는 Tau 단백질이, non-neurological tissue에서는 AHNAK 단백질이 cPLA2의 산물인 arachidonic acid (AA)가 존재하는 조건에서 PLC-γ를 활성화시키는 것이 알려짐으로써 생체내에서 cPLA2와 PLC-γ사이의 밀접한 관계 (cross-talk)가 제시되었다. 더 나아가 PLC-γ와 AHNAK 단백질은 AA가 존재하는 조건에서만 물리적으로 결합하는 것이 증명되었다. 이러한 세포내 기능을 갖는 AHNAK 단백질은 PKC에 의해 인산화 되어 세포막쪽으로 이동되는 것이 밝혀졌는데 인산화된 AHNAK이 PLC-r에는 어떠한 영향을 주는지에 대해 조사해 보았다. PKC에 의한 PLC-γ의 인산화는 PLC 활성에 negative feedback 효과를 보이는 것으로 알려져 있다. 본 논문의 실험에서는 PKC에 의하여 인산화된 AHNAK 단백질이 PLC-γ의 활성을 증가시키며, 인산화된 AHNAK이 AA 존재 하에서 PLC-γ와 더 강한 결합을 하는 것을 볼수 있었다. 또한 AHNAK에 의한 PLC-γ의 활성화를 in vivo 에서도 확인하였다. PLC-γ 가 과발현되어 있는 NIH 3T3 2.2 세포에 AHNAK을 transfection 한 후 PMA자극을 주니 AHNAK을 넣지 않은 세포에 자극을 주었을 때보다 PLC의 활성이 상대적으로 높아졌다. In vivo에서도 PKC에 의한 자극으로 AHNAK 단백질이 PLC-γ를 활성화시킴을 확인하였다. 이러한 결과들은 PLC-γ에 대한 PKC의 효과가 AHNAK을 통해 positive feedback으로 작용함을 보여주고 있다. ; Several lines of evidence suggested that phospholipase C-γ,(PLC-γ) is activated either by tau in a neuronal cell-specific microtubule-associated protein, or by AHNAK protein in non-neuronal tissues in the presence of arachidonic acid. AHNAK was initially identified by Bishop and colleagues as the product of a gene whose expression is repressed in human neuroblastomas and several other types of tumor cell lines and named AHNAK to convey its exceptional size. AHNAK was also independently isolated as a 680 kDa protein uniquely localized to the desmosomes of bovine muzzle epidermal cells and termed desmoyokin. The genomic sequence predicts a length of 5643 amino acids for AHNAK. The central region of AHNAK is comprised of approximately 30 repeated motifs. The repeated unit is 128 amino acids in length, but some of the units are less than 128 residues. The 128-residue motifs are on average approximately 80% identical to each other with respect to amino acid sequence. Examination of the amino acid sequence within the repeated unit reveals a number of recurrences of hepta- (or octa-) sequence (D/E)ΦΦΦK(A/G)P(K), where Φ represents a hydrophobic residue, Φ represents a hydrophilic residue, and other letters correspond to conventional one letter representation of amino acid residues. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-Υ1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-Υ1 activation. Concerted action of AHNAK and AA stimulated PLC-Υ1 activity. Furthermore, the AA was to promote a physical interaction between AHNAK and PLC-Υ1. Given that and AHNAK are known to be phosphoproteins, phosphorylation may play a role in regulation of their function as PLC-Υ1 activators. In keratinocytes, AHNAK was mainly in the cytoplasm when cells were kept in low Ca^2+ medium, but translocated to the plasma membranes with a concomitant increase in the degree of phosphorylation upon an increase in extracellular Ca^2+ concentrations or treatment with phorbol ester. The level of expression as well as the state of phosphorylation of the protein in several transformed cells was altered during growth and differentiation. To investigate the effect of phosphorylation of AHNAK on PLC-Υ1 activity, PKC-phosphorylated AHNAK proteins are engaged. PKC-phosphorylated AHNAK proteins more activated PLC-Υ1 activity than unphosphorylated AHNAK. To verify the function of AHNAK in vivo, we determined total IP in cells in response to PMA. Stimulation of AHNAK-transfected NIH3T3 2.2 cells with PMA resulted in 40% increase of total EP than NIH3T3 2.2 cells. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-Υ1 activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation. Moreover, AHNAK protein can mediate positive feedback loop between PKC and PLC-Υ1 upon stimulation of PKC.
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