臺灣博碩士論文加值系統

　　一種被命名為自我磷酸化活化激脢的絲氨酸息寧氨酸激脢已從神經組織及非神經組織中純化鑑定出來，這種激脢以非活化狀態存在，然而它在鎂離子及腺核甘三磷酸存在下可被活化，這個活化過程包括了分子內的自我磷酸化，此活化後的激脢變得能對幾種包括核鹼性蛋白、肝醣合成脢、腦髓鞘鹼性蛋白、和去磷酸脢等受質作用，因此自我磷酸化活化激脢可能對廣泛的細胞功能有調控的功能。
　　為了尋找證實自我磷酸化活化激脢之受質氨基酸序列的共同特徵，我們使用了一種純化出來的蛋白質腦髓鞘鹼性蛋白以及一系列的胜月太為其受質。在經由變向薄層色層分析、酸磷氨基酸種類分析、高效能液態層析分析、愛德曼降階分析和直接勝胜□分析，發現腦髓鞘性蛋白有三個主要及二個次要的磷酸化位置可被自我磷酸化活化激脢作用，在比較主要磷酸化位置附近的序列，我們猜測：精氨酸 - x - (x) - 絲氨酸／息寧氨酸 - （中性氨基酸）3 - （含氫氧基之氨基酸）是自我磷酸化活化激脢受質氨基酸序列的共同特徵。
　　為了進一步證實這可能的受質氨基酸序列共同特徵，我們使用了一段根據肝醣合成脢第三位置之序列而含十七個氨基酸的胜月太一肝醣合成脢汰三為測試模式，此勝□已經我們證實是自我磷酸化活化激脢的極佳受質。肝醣合成脢勝□三被縮短或修改成幾種不同長度或氨基酸的勝□並用以測試自我磷酸化活化激脢對它們的活性。所有的結果顯示：對自我磷酸化活化激脢而言，精氨酸 - x - （x） - 絲氨酸／息寧氨酸是最低現度的受質辨認特徵；精氨酸 - x - （x） - 絲氨酸／息寧氨酸 -（中性氨基酸）3 - （絲氨酸／息寧氨酸）是最佳的受質辨認特徵；另外，所有結果顯示：精氨酸 - x -（x）-絲氨酸／息寧氨酸 -（中性氨基酸）3 - （含氫氧基之氨基酸）-是自我磷酸化活化激脢受質氨基酸序列的共同特徵。

　　In a previous report (Yang et al., (1987a) J. Biol. Chem. 262, 7034 - 7040), a cyclic AMP and calcium - independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation - dependent protein kinase (auto - kinase) were further determined by two - dimensional electrophoresis / thin - layer chromatography, phosphoamino acid analysis, high performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation and direct peptide sequencing. Auto - kinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C18 - reversed phase high pefformance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS (p) WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio ~ 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser115, one of the in vivo phosphorylation sites in MBP, as the major site phosphorylated by auto - kinase, implicating a physiologically relevant role of auto - kinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT (p) GILDSLGR (molar ratio ~ 0.9) and TT (p) HYGSLPQK (molar ratio ~ 0.8) as the major phosphorylation sites sequences in 32p - MBP phosphorylated by auto - kinase, further indicating that - Arg - X - Ser / Thr - (neutral amino acid) 3 - (amino acid containing hydroxyl group such as Ser / Glu / Asp) - (neutral amino acid) 2 - may represent a unique consensus sequence motif specifically recognized by this autophosphorylationdependent multisubstrate / multifunctional protein kinase in the brain.
　　To further confirm the consensus sequence motif for this autophosphorylation-dependent protein serine / threonine kinase, we have tested several synthetic peptides. The well - established protein serine / threonine kinases such as cAMP - dependent protein kinase (PKA), Ca2 + / calmodulin - dependent protein kinase (CaM - kinase) and protein kinase C (PKC) were found to be inactive towards phosphorylation of synitde - 3 (RPRPASVPPS - PSLSRHA), which turned out to be an excellent substrate only for auto - kinase, indicating that syntide - 3 is a specific substrate for auto - kinase, indicating that syntide - 3 is a specific substrate for auto - kinase. Modification of syntide - 3 to become RPRPASVPPS / T did not affect the activity of auto - kinase. By contrast, auto - kinase. rather or almost inactive when the peptide was modified to become RPRPASVPPA / G / F / K / R / D / E / Y, indicating that amino acid number 10 in syntide - 3 is crucial to the sequence motif recognized by auto - kinase. Taken together, the results provide initial evidence that - Arg - X - (X) - Ser / Thr - X3 - (amino acid containing hydroxyl group) - may represent a unique consensus sequence motif specifically recognized by autophosphorylation - dependent protein kinase, a new family of multisubstrate / multifunctional protein serine / threonine kinase.