可對蔗糖合成瓷絲胺酸基進行多重磷酸化修飾之水稻錳离子依賴型蛋白質激瓷之檢定

Identification of Rice manganese-dependent Protein Kinases that Phosphorylate Sucrose Synthase at Multiple Serine Residues

蔡承佳(Zheng-Chia Tsaiand) ； 王愛玉(Ai-Yu Wang)

《Botanical Bulletin of Academia Sinica》 44卷2期 (2003/04) Pp. 141-150

https://doi.org/10.7016/BBAS.200304.0141

摘要

台農67號水稻白化苗中之蔗糖合成瓷(sucrose synthase)於細胞中及試管中皆可受到蛋白質激瓷的磷化修飾。由水稻白化苗中部份純化得四種可對重組水稻蔗糖合成瓷1（簡稱RSuS1）進行磷酸化修飾之錳離子依賴型蛋白質激瓷。這四種激瓷（分別命名為RPK1、RPK2、PRK3、RPK4）均為單元體形式之酵素，分子量分別為34、57、30及30kDa。由磷酸化胺基酸分析結果得知四種RPKs對重組RSuS1之磷酸化修飾是發生在絲胺酸基。以胰蛋白瓷(trypsin)水解磷酸化之重組RSuS1得到之胜肽片段，進行逆相高效層析(RP-HPLC)及電儷遊離質譜儀(ESI-MS)分析，得知13-LHSVR-17及168-HLSSK-172兩條胜肽片段中之絲胺酸可能為磷酸化修飾之目標位置。為了證實此發現，進一步將S15A、S170A、及S15A/S170A等重組RSuS1突變蛋白質由大腸悍菌純化出，並以此四種部分純化之蛋白質激瓷進行磷酸化反應。結果顯示，Ser15及Ser170為RPK1、RPK2及RPK3之目標基，且以Ser15為主要磷酸化位置;雙突變蛋白質S15A/S170A亦可觀察到磷酸化修飾現象，表示在重組RSuS1中，磷酸化非僅發生於此二絲胺酸基。RPK4對突變蛋白質S15A及S15A/S170A之磷酸化修飾無法被偵測到，顯示此酵素僅可能作用於Ser15。

關鍵字

錳离子依賴型激瓷 ； 水稻 ； 蔗糖合成瓷

並列摘要

Sucrose synthase from the etiolated seedlings of flee (Oryza sativa L. cv. Tainung 67) was phosphorylated both in vivo and in vitro. Four protein kinases that phosphorylated recombinant rice sucrose synthase1 (RSuS1) in a Mn(Superscript 2+)-dependent manner were partially purified and characterized from etiolated rice seedlings. These four kinases, designated as RPK1, RPK2, RPK3 and RPK4, are monomeric enzymes with apparent molecular masses of 34 kDa, 57 kDa, 30 kDa, and 30 kDa, respectively. Phosphoamino acid analysis of the (superscript 32)P-labeled phosphorylated recombinant RSuS1 indicated that it was phosphorylated at serine residues by these four RPKs. RP-HPLC/ESI-MS analysis of the tryptic peptides of phosphorylated RSuS suggested that the serine residues in the tryptic peptides 13-LHSVR-17 and 168-HLSSK-172 were the target residues for phosphorylation. For confirmation of this finding, mutant recombinant RSuS1, S15A, S170A and S15A/S170A, were purified and subjected to phosphorylation by the four partially purified kinases. The results showed that both Ser15 and Ser170 residues were target residues for RPK1, PRK2 and PRK3 and Ser15 was the major phosphorylation site in RSuS1. Phosphorylation of RSuS1 may not occur exclusively at these two sites since weak phosphorylation of the double mutant protein S15A/S 170A was also observed. Phosphorylation of the mutant S15A and S 15A/S17OA by RPK4 was undetectable, indicating that Ser15 was the only target residue for this kinase.

並列關鍵字

Manganese-dependent protein kinases ； Rice ； Sucrose synthase