Harper J F, Huang J F, Lloyd S J
Department of Cell Biology, Scripps Research Institute, La Jolla, California 92037.
Biochemistry. 1994 Jun 14;33(23):7267-77. doi: 10.1021/bi00189a031.
CDPKs are a family of calcium (Ca2+)-dependent protein kinases which are defined by a carboxyl-terminal calmodulin-like domain. Mutational analysis indicates that the junction domain, which joins the kinase and calmodulin-like domains, contains an autoinhibitor. CDPK isoform AK1 from Arabidopsis was expressed in Escherichia coli as a fusion protein sandwiched between glutathione S-transferase and six consecutive histidines at the N- and C-terminal ends, respectively. This fusion, called AK1-6H, was purified and displayed kinase activity which was stimulated up to 127-fold by Ca2+, with a typical specific activity of 2000 nmol min-1 mg-1, using syntide-2 as peptide substrate. A truncation which deletes the calmodulin-like domain, as in mutant delta C-6H, disrupts Ca2+ activation and leaves the enzyme with a basal level of activity. Delta C-6H could be activated 87-fold by preincubation with a purified polyclonal IgG which was raised against a junction domain fusion. A further deletion of the junction domain, as in mutant delta JC, results in a constitutively active enzyme. This indicates that the junction domain in delta C-6H can function as an autoinhibitor. Its function as an autoinhibitor in a full-length enzyme was confirmed by site-specific mutagenesis, as shown by mutant KJM23-6H, which had a six-residue substitution in the junction domain between A422 and A432. Both delta JC and KJM23-6H encoded Ca(2+)-independent enzymes which had specific activities greater than 70% that of a fully active AK1-6H and displayed equivalent Km values for ATP and syntide-2. Inhibition studies on delta JC, using peptides based on the autoinhibitory domains of Ca2+/calmodulin-dependent protein kinases, are consistent with a model where the junction domain contains a similar pseudosubstrate-type autoinhibitor.
钙依赖蛋白激酶（CDPKs）是一类钙（Ca2+）依赖的蛋白激酶，其由羧基末端类钙调蛋白结构域定义。突变分析表明，连接激酶结构域和类钙调蛋白结构域的连接结构域含有一个自身抑制剂。拟南芥的CDPK同工型AK1在大肠杆菌中作为融合蛋白表达，该融合蛋白分别在N端和C端夹在谷胱甘肽S-转移酶和六个连续的组氨酸之间。这种融合蛋白称为AK1-6H，经过纯化后显示出激酶活性，以合成肽-2为肽底物时，其活性被Ca2+刺激高达127倍，典型的比活性为2000 nmol min-1 mg-1。如在突变体delta C-6H中那样，缺失类钙调蛋白结构域的截短形式会破坏Ca2+激活，并使酶具有基础水平的活性。通过与针对连接结构域融合蛋白产生的纯化多克隆IgG预孵育，delta C-6H可被激活87倍。如在突变体delta JC中那样，进一步缺失连接结构域会导致一种组成型活性酶。这表明delta C-6H中的连接结构域可作为自身抑制剂发挥作用。通过定点诱变证实了其在全长酶中作为自身抑制剂的功能，如突变体KJM23-6H所示，该突变体在A422和A432之间的连接结构域中有六个残基的替换。delta JC和KJM23-6H都编码不依赖Ca(2+)的酶，其比活性大于完全活性的AK1-6H的70%，并且对ATP和合成肽-2显示出相当的Km值。使用基于Ca2+/钙调蛋白依赖蛋白激酶自身抑制结构域的肽对delta JC进行的抑制研究与一个模型一致，该模型认为连接结构域含有类似假底物型的自身抑制剂。
