Stokoe D, Caudwell B, Cohen P T, Cohen P
Department of Biochemistry, University of Dundee, Scotland, UK.
Biochem J. 1993 Dec 15;296 ( Pt 3)(Pt 3):843-9. doi: 10.1042/bj2960843.
The substrate specificity of mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MAPKAP kinase-2) was investigated by using synthetic peptides related to the N-terminus of glycogen synthase. The minimum sequence required for efficient phosphorylation was found to be Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa, where Hyd is a bulky hydrophobic residue (Phe > Leu > Val >> Ala), and the peptide Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala was phosphorylated with a Km of 9.3 microM and Vmax. of 10 mumol/min per mg. MAPKAP kinase-1 (a homologue of ribosomal protein S6 kinase) also requires an arginine three residues N-terminal to the serine (position n-3), but not a hydrophobic residue at position n-5. Neither MAPKAP kinase-1 nor MAPKAP kinase-2 could tolerate a proline residue at position n + 1, indicating that their specificities do not overlap with that of MAP kinase. The specificity of calmodulin-dependent protein kinase-II resembled that of MAPKAP kinase-2, except that it could tolerate replacement of the arginine by a lysine and the phosphorylation-site serine by a threonine residue. Partial cDNAs encoding MAPKAP kinase-2 were isolated from rabbit and human skeletal muscle and human teratocarcinoma libraries, and Northern-blotting experiments revealed a single 3.3 kb mRNA transcript present at similar levels in six human tissues examined. The catalytic domain was most similar (35-40% identity) to calmodulin-dependent protein kinases II and IV, phosphorylase kinase, putative serine kinase H1 and the C-terminal domain of MAPKAP kinase-1, which form one branch of the protein kinase phylogenetic tree. The sequence N-terminal to the catalytic domain is proline-rich and contains two putative SH3-binding sites. The threonine residue phosphorylated by MAP kinase lies immediately C-terminal to the catalytic domain and is followed by a nuclear localization signal, Lys-Lys-(Xaa)10-Lys-Arg-Arg-Lys-Lys, near the C-terminus.
通过使用与糖原合酶N端相关的合成肽，研究了丝裂原活化蛋白（MAP）激酶激活的蛋白激酶2（MAPKAP激酶2）的底物特异性。发现有效磷酸化所需的最小序列为Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa，其中Hyd是一个大的疏水残基（苯丙氨酸>亮氨酸>缬氨酸>>丙氨酸），肽Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala被磷酸化，其Km为9.3微摩尔，Vmax为每毫克10微摩尔/分钟。MAPKAP激酶1（核糖体蛋白S6激酶的同源物）也需要在丝氨酸N端三个残基处有一个精氨酸（n-3位置），但在n-5位置不需要疏水残基。MAPKAP激酶1和MAPKAP激酶2在n + 1位置都不能耐受脯氨酸残基，这表明它们的特异性与MAP激酶的特异性不重叠。钙调蛋白依赖性蛋白激酶II的特异性与MAPKAP激酶2相似，只是它可以耐受赖氨酸取代精氨酸以及苏氨酸残基取代磷酸化位点的丝氨酸。从兔和人骨骼肌以及人畸胎瘤文库中分离出编码MAPKAP激酶2的部分cDNA，Northern印迹实验显示在所检测的六种人体组织中存在单一的3.3 kb mRNA转录本，且水平相似。催化结构域与钙调蛋白依赖性蛋白激酶II和IV、磷酸化酶激酶、推定的丝氨酸激酶H1以及MAPKAP激酶1的C端结构域最相似（同一性为35-40%），它们构成了蛋白激酶系统发育树的一个分支。催化结构域N端的序列富含脯氨酸，并包含两个推定的SH3结合位点。被MAP激酶磷酸化的苏氨酸残基紧邻催化结构域的C端，随后在C端附近有一个核定位信号Lys-Lys-(Xaa)10-Lys-Arg-Arg-Lys-Lys。
