BETA-ADRENERGIC-RECEPTOR KINASE - IDENTIFICATION OF A NOVEL PROTEIN-KINASE THAT PHOSPHORYLATES THE AGONIST-OCCUPIED FORM OF THE RECEPTOR
BETA-ADRENERGIC-RECEPTOR KINASE - IDENTIFICATION OF A NOVEL PROTEIN-KINASE THAT PHOSPHORYLATES THE AGONIST-OCCUPIED FORM OF THE RECEPTOR
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DOI：
10.1073/pnas.83.9.2797
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发表时间：
1986-05-01
影响因子：
11.1
通讯作者：
LEFKOWITZ, RJ
中科院分区：
文献类型：
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作者：
BENOVIC, JL;STRASSER, RH;LEFKOWITZ, RJ
激动剂促进的腺苷酸环化酶脱敏与哺乳动物、鸟类和两栖类细胞中β -肾上腺素能受体的磷酸化密切相关。然而，参与受体磷酸化的蛋白激酶的性质在很大程度上仍然未知。我们在此报道一种能够磷酸化纯化的β -肾上腺素能受体的激动剂结合形式的蛋白激酶的鉴定和部分纯化。该酶是从裂解的kin⁻细胞（S49淋巴瘤细胞的一种突变体，缺乏有功能的环腺苷酸依赖性蛋白激酶）高速离心后的上清液部分制备的。β -激动剂异丙肾上腺素使该激酶对受体的磷酸化作用增加5 - 10倍，而这种作用可被拮抗剂阿普洛尔阻断。通过分子筛高效液相色谱和DEAE - Sephacel对kin⁻上清液进行分级分离，得到一种纯化了50 - 100倍的β -肾上腺素能受体激酶制剂，该制剂基本上没有其他蛋白激酶活性。该激酶活性对环腺苷酸、环鸟苷酸、环腺苷酸依赖性激酶抑制剂、Ca²⁺ -钙调蛋白、Ca²⁺ -磷脂和佛波酯不敏感，并且不磷酸化一般的激酶底物，如酪蛋白和组蛋白。磷酸似乎仅仅掺入丝氨酸残基。这种新型的不依赖环腺苷酸的激酶的存在，它优先磷酸化β -肾上腺素能受体的激动剂结合形式，提示了一种可能解释腺苷酸环化酶脱敏的同源或激动剂特异性形式的机制。它还提示了一种调节受体功能的一般机制，即只有受体的激动剂结合或“活性”形式才是诱导共价修饰的酶的底物。
Agonist-promoted desensitization of adenylate cyclase is intimately associated with phosphorylation of the .beta.-adrenergic receptor in mammalian, avian, and amphibian cells. However, the nature of the protein kinase(s) involved in receptor phosphorylation remains largely unknown. We report here the identification and partial purification of a protein kinase capable of phosphorylating the agonist-occupied form of the purified .beta.-adrenergic receptor. The enzyme is prepared from a supernatant fraction from high-speed centrifugation of lysed kin- cells, a mutant of S49 lymphoma cells that lacks a functional cAMP-dependent protein kinase. The .beta.-agonist isoproterenol induces a 5- to 10-fold increase in receptor phosphorylation by this kinase, which is blocked by the antagonist alprenolol. Fractionation of the kin- supernatant on molecular-sieve HPLC and DEAE-Sephacel results in a 50- to 100-fold purified .beta.-adrenergic receptor kinase preparation that is largely devoid of other protein kinase activities. The kinase activity is insensitive to cAMP, cGMP, cAMP-dependent kinase inhibitor, Ca2+-calmodulin, Ca2+-phospholipid, and phorbol esters and does not phosphorylate general kinase substrates such as casein and histones. Phosphate appears to be incorporated solely into serine residues. The existence of this novel cAMP-independent kinase, which preferentially phosphorylates the agonist-occupied form of the .beta.-adrenergic receptor, suggests a mechanism that may explain the homologous or agonist-specific form of adenylate cyclase desensitization. It also suggests a general mechanism for regulation of receptor function in which only the agonist-occupied or "active" form of the receptor is a substrate for enzymes inducing covalent modification.