Regulation of the M2 pyruvate kinase through direct interaction with the ITAM of the FcεRI gamma chain = FcεRI gamma chain의 ITAM과의 직접적인 상호작용에 의해 이루어지는 M2 pyruvate kinase 활성조절에 관한 연구

국문 초록 (Abstract)

본 연구는 고 친화도 IgE 수용체인 FcεRI γ chain의 신호전달체계를 규명하기 위한 일환으로 FcεRI과 puruvate kinase 간에 이루어지는 작용이 갖는 기능적 의미와 그 조절관계를 분자생물학적으로 밝히고자 시도되었다. 그 결과 FcεRI의 γ chain과 작용하는 pyruvate kinase는 M2 type이며 특히 γ chain의 ITAM (immunoreceptor tyrosine based activation motif)이 pyruvate kinase 와의 작용에 있어서 결합 부분으로 제공됨을 yeast two-hybrid system을 통해 알 수 있었다. FcεRI은 pyruvate kinase의 활성을 즉시, 그리고 지속적으로 저해 시킴과 동시에 pyruvate kinase의 tyrosine과 serine 잔기의 인산화를 유발했다. 그리고 이러한 pyrute kinase의 활성조절에 있어서 PI 3-kinase와 protein kinase C가 관여됨을 protein kinase 저해제 (wortmanin, PMA, Go¨6 6976 and Go¨ 6983)를 이용해 밝혔다. 이러한 결과들은 FcεRl 활성화됨에 따라 pyruvate kinase의 인산화가 유발되어 효소활성이 감소되고 나아가 mast cell의 탈 과립이 저해되는 이른바 FcεRI의 negative control mechanism으로 pyruvate kinase가 작용함을 보여 준다.

다국어 초록 (Multilingual Abstract)

In this study, the functional significance and regulation of the pyruvate kinase was characterized as a part of identifying signal transduction of high affinity IgE receptor (Fc ε RI). The subtype of pyruvate kinase which interacts with γ chain of Fc ε RI was revealed to be M2 type. Series of constructs were prepared for the cytoplasmic loop of γ chain, and tested for the interaction with M2 type pyruvate kinase in yeast. The immunoreceptor tyrosine based activation motif (ITAM) of γ chain was designated to be the domain required for the interaction of two proteins. The cross-linking of Fc ε RI caused an immediate and sustained inhibition of enzyme activity and the phosphorylation of pyruvate kinase on tyrosine and serine residues. It was also demonstrated that both PI 3-kinase and protein kinase C were involved in the IgE-mediated regulation of the pyruvate kinase using PI 3-kinase inhibitor (wortmannin) and protein kinase C inhibitors (phorbol-12-myristate-13 acetate, Go¨ 6976 and Go¨ 6983). As a result, the inhibition of the pyruvate kinase by changes in phosphorylation following the stimulation of Fc ε RI might work as a regulator for negative control mechanism in the intracellular signaling pathway of Fc ε RI.

목차 (Table of Contents)

• 목차
• CONTENTS = i
• LIST OF FIGURES = iii
• LIST OF TABLE = iv
• LIST OF ABBREVIATIONS = v
• ABSTRACT (in English) = vii
• INTRODUCTION = 1
• MATERIALS AND METHODS = 4
• I. Materials = 4
• 1. Yeast strain and vectors = 4
• 2. Cell line = 4
• 3. Antibodies = 4
• 4. Chemicals = 5
• II. Methods = 6
• 1. Polymerase chain reaction = 6
• 2. Preparation of DNA constructs and yeast transformation = 6
• 3. Hexosaminidase release from RBL-2H3 cells = 6
• 4. Enzymatic assay of pyruvate kinase = 7
• 5. Phosphorylation studies = 8
• RESULTS AND DISCUSSION = 10
• 1. Identification of the subtype of pyruvate kinase that interacts with Fc ε RI = 10
• 2. Identification of binding site on the γ chain of Fc ε RI = 13
• 3. The time-course of degranulation and pyruvate kinase activity after the stimulation of Fc ε RI in RBL-2H3 cells = 15
• 4. Phoshorylation of pyruvate kinase by the activation of Fc ε RI = 18
• 5. Effects of protein kinases on activity and phosphorylation of pyruvate kinase = 20
• REFERENCES = 28
• ABSTRACT (in Korean) = 35