合成肽已用于定义减数分裂激活的髓磷脂碱性蛋白(MBP)激酶(p44mpk)的底物识别的共有氨基酸序列,从成熟的海星卵母细胞中纯化。该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)具有许多特性。最近,牛MBP的胰蛋白酶片段KNIVTPRTPRTPPPSQGK中的Thr-97被p44mpk鉴定为磷酸化的主要位点(Sanghera,J.S.,Aebersold,R、莫里森,H.D.,Bures,E.J.,和Pelech,S、L.(1990)FEBSLett。273、223-226)。以该序列为模型的合成肽显示,可磷酸化苏氨酸(或丝氨酸)残基的脯氨酸残基C末端(1位)的存在对于p44mpk的识别至关重要。虽然不是必需的,位于-2位置的脯氨酸残基增强了肽磷酸化的Vmax。基本,酸性,和非极性残基在-1位同样耐受。-3位氨基酸残基的存在也增加了肽磷酸化。因此,p44mpk磷酸化的最佳共有序列定义为Pro-X-(Ser/Thr)-Pro,其中X是可变氨基酸残基,但理想情况下不是Pro。包含该序列的肽被p44mpk磷酸化,Vmax值接近1μmol。min-1.mg-1,表观Km值为约1mM)。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3mM)。超过40种不同的蛋白激酶含有Pro-X-(Ser/Thr)-Pro序列,包括胰岛素和表皮生长因子的人类受体,和人类原癌基因abl编码的激酶,neu,和raf-1,以及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中也鉴定出多个推定位点,人类tau蛋白,以及果蝇myb蛋白和RNA聚合酶II。
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.