Kornstein L B, Gaiso M L, Hammell R L, Bartelt D C
Department of Biological Sciences, St. John's University Jamaica, NY 11439.
Gene. 1992 Apr 1;113(1):75-82. doi: 10.1016/0378-1119(92)90671-b.
A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.
利用针对该酶的单特异性多克隆抗体，通过免疫筛选从λZAP表达文库中分离出编码构巢曲霉钙调蛋白依赖性多功能蛋白激酶（ACMPK）的部分cDNA。测定了该cDNA（CMKa）两条链的序列。推导的氨基酸（aa）序列包含丝氨酸/苏氨酸蛋白激酶中发现的所有11个共有结构域[汉克斯等人，《科学》241（1988）42 - 52]，以及一个假定的钙调蛋白结合结构域。该cDNA含有一个内含子，缺少读码框内的起始密码子，且未进行多聚腺苷酸化。随后，使用第一个克隆的限制性片段作为探针，从构巢曲霉cDNA的λgt10文库中分离出CMKa的全长拷贝。它含有读码框内的起始密码子、一个1242 bp的开放阅读框（ORF），并进行了多聚腺苷酸化。该ORF编码一个由414个氨基酸残基组成的蛋白质，分子量为46,895，等电点pI = 6.4。这些值与天然酶的观察值[巴特尔特等人，《美国国家科学院院刊》85（1988）3279 - 3283]非常一致。当进行比对以优化同源性时，ACMPK预测的氨基酸序列中有29%与大鼠脑钙调蛋白依赖性蛋白激酶II的α亚基相同。ACMPK与最近从酿酒酵母中克隆的两种Ca²⁺/钙调蛋白依赖性蛋白激酶YCMK1和YCMK2的氨基酸序列分别具有40%和44%的同一性[保施等人，《欧洲分子生物学组织杂志》10（1991）1511 - 1522]。基因组DNA限制性酶切的Southern分析结果表明，ACMPK由单拷贝基因编码。
