Clark-Lewis I, Sanghera J S, Pelech S L
Biomedical Research Centre, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1991 Aug 15;266(23):15180-4.
Synthetic peptides have been used to define the consensus amino acid sequence for substrate recognition by the meiosis-activated myelin basic protein (MBP) kinase (p44mpk), which was purified from maturing sea star oocytes. This protein kinase shares many properties with the mitogen-activated microtubule-associated protein-2 kinase (p42mapk) in vertebrates. Recently, Thr-97 in the tryptic fragment KNIVTPRTPPPSQGK of bovine MBP was identified as the major site of phosphorylation by p44mpk (Sanghera, J. S., Aebersold, R., Morrison, H. D., Bures, E. J., and Pelech, S. L. (1990) FEBS Lett. 273, 223-226). Synthetic peptides modeled after this sequence revealed that the presence of a proline residue C-terminal (+1 position) to the phosphorylatable threonine (or serine) residue was critical for recognition by p44mpk. Although not essential, a proline residue located at the -2 position enhanced the Vmax of peptide phosphorylation. Basic, acidic, and non-polar residues were equally tolerated at the -1 position. The presence of an amino acid residue at position -3 also increased peptide phosphorylation. Thus, the optimum consensus sequence for phosphorylation by p44mpk was defined as Pro-X-(Ser/Thr)-Pro, where X is a variable amino acid residue, but ideally not a Pro. Peptides that included this sequence were phosphorylated by p44mpk with Vmax values approaching 1 mumol.min-1.mg-1 and with apparent Km values of approximately 1 mM). Pseudosubstrate peptides in which the phosphorylatable residue was replaced by valine or alanine were weak inhibitors of p44mpk (apparent Ki values of approximately 3 mM). Over 40 distinct protein kinases contain Pro-X-(Ser/Thr)-Pro sequences including the human receptors for insulin and epidermal growth factor, and kinases encoded by the human proto-oncogenes abl, neu, and raf-1, and Schizosaccharomyces pombe cell cycle control genes ran-1 and wee-1. Multiple putative sites were also identified in rat microtubule-associated protein-2, human retinoblastoma protein, human tau protein, and Drosophila myb protein and RNA polymerase II.
合成肽已被用于确定减数分裂激活的髓鞘碱性蛋白（MBP）激酶（p44mpk）识别底物的共有氨基酸序列，该激酶是从成熟的海星卵母细胞中纯化出来的。这种蛋白激酶与脊椎动物中的丝裂原激活的微管相关蛋白2激酶（p42mapk）具有许多共同特性。最近，牛MBP胰蛋白酶片段KNIVTPRTPPPSQGK中的苏氨酸97被确定为p44mpk磷酸化的主要位点（桑赫拉，J.S.，埃伯索尔德，R.，莫里森，H.D.，布雷斯，E.J.，和佩莱奇，S.L.（1990年）《欧洲生物化学学会联合会快报》273，223 - 226）。以该序列为模型的合成肽表明，在可磷酸化的苏氨酸（或丝氨酸）残基的C末端（+1位）存在脯氨酸残基对于p44mpk的识别至关重要。虽然不是必需的，但位于 -2位的脯氨酸残基提高了肽磷酸化的Vmax。在 -1位，碱性、酸性和非极性残基同样可以被容忍。在 -3位存在氨基酸残基也增加了肽的磷酸化。因此，p44mpk磷酸化的最佳共有序列被定义为Pro - X -（Ser/Thr）- Pro，其中X是可变氨基酸残基，但理想情况下不是脯氨酸。包含该序列的肽被p44mpk磷酸化，Vmax值接近1 μmol·min⁻¹·mg⁻¹，表观Km值约为1 mM。其中可磷酸化残基被缬氨酸或丙氨酸取代的假底物肽是p44mpk的弱抑制剂（表观Ki值约为3 mM）。超过40种不同的蛋白激酶包含Pro - X -（Ser/Thr）- Pro序列，包括人胰岛素和表皮生长因子受体，以及人原癌基因abl、neu和raf - 1编码的激酶，还有粟酒裂殖酵母细胞周期控制基因ran - 1和wee - 1。在大鼠微管相关蛋白2、人视网膜母细胞瘤蛋白、人tau蛋白和果蝇myb蛋白以及RNA聚合酶II中也鉴定出了多个假定位点。
