Substrate recognition by Arg/Pro‐rich insert domain in calcium/calmodulin‐dependent protein kinase kinase for target protein kinases
Substrate recognition by Arg/Pro‐rich insert domain in calcium/calmodulin‐dependent protein kinase kinase for target protein kinases
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DOI：
10.1111/febs.16467
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发表时间：
2022-05
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通讯作者：
Riku Kaneshige;Satomi Ohtsuka;Yuhei Harada;Issei Kawamata;M. Magari;N. Kanayama;N. Hatano;H. Sakagami;H. Tokumitsu
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作者：
Riku Kaneshige;Satomi Ohtsuka;Yuhei Harada;Issei Kawamata;M. Magari;N. Kanayama;N. Hatano;H. Sakagami;H. Tokumitsu
钙/钙调蛋白依赖性蛋白激酶激酶（CaMKK）通过磷酸化激活环中的Thr残基来激活CaMKI、CaMKIV、蛋白激酶B/Akt和AMP激活的蛋白激酶（AMPK），以介导各种Ca 2+信号通路。表达缺乏Arg/Pro丰富插入结构域（RP-结构域）序列的CaMKKα和CaMKKβ的哺乳动物细胞显示AMPKα、CaMKIα和CaMKIV的磷酸化受损，而CaMKK突变体的自磷酸化活性保持完整，与野生型CaMKK相似。肝激酶B1（LKB 1，一种AMPK激酶）与STRAD和MO 25复合，不能磷酸化CaMKIα和CaMKIV;然而，在激酶亚结构域II和III之间插入CaMKKα和CaMKKβ RP-结构域序列的突变体LKB 1在体外和转染的培养细胞中获得了CaMKIα和CaMKIV磷酸化活性。此外，在转染细胞中，离子霉素诱导的血凝素（HA）-CaMKIα在Thr 177、HA-CaMKIV在Thr 196和HA-AMPKα在Thr 172的磷酸化被CaMKK亚型的激酶死亡突变体共转染显著抑制，但这些显性负效应被RP缺失突变体消除，表明功能丧失的CaMKK突变体对底物激酶的螯合需要RP结构域。下拉实验证实了这一点，该实验表明CaMKKα和CaMKKβ的显性阴性突变体与靶激酶相互作用，但不与RP缺失突变体相互作用。总之，这些结果清楚地表明，两种CaMKK亚型都需要RP-结构域识别下游激酶，以与其激活环中的Thr残基相互作用并磷酸化Thr残基。因此，RP-结构域可能是特异性CaMKK抑制剂的有希望的靶点。
Calcium/calmodulin‐dependent protein kinase kinases (CaMKKs) activate CaMKI, CaMKIV, protein kinase B/Akt, and AMP‐activated protein kinase (AMPK) by phosphorylating Thr residues in activation loops to mediate various Ca2+‐signaling pathways. Mammalian cells expressing CaMKKα and CaMKKβ lacking Arg/Pro‐rich insert domain (RP‐domain) sequences showed impaired phosphorylation of AMPKα, CaMKIα, and CaMKIV, whereas the autophosphorylation activities of CaMKK mutants remained intact and were similar to those of wild‐type CaMKKs. Liver kinase B1 (LKB1, an AMPK kinase) complexed with STRAD and MO25 and was unable to phosphorylate CaMKIα and CaMKIV; however, mutant LKB1 with the RP‐domain sequences of CaMKKα and CaMKKβ inserted between kinase subdomains II and III acquired CaMKIα and CaMKIV phosphorylating activity in vitro and in transfected cultured cells. Furthermore, ionomycin‐induced phosphorylation of hemagglutinin (HA)–CaMKIα at Thr177, HA–CaMKIV at Thr196, and HA–AMPKα at Thr172 in transfected cells was significantly suppressed by cotransfection of kinase‐dead mutants of CaMKK isoforms, but these dominant‐negative effects were abrogated with RP‐deletion mutants, suggesting that sequestration of substrate kinases by loss‐of‐function CaMKK mutants requires the RP‐domain. This was confirmed by pulldown experiments that showed that dominant‐negative mutants of CaMKKα and CaMKKβ interact with target kinases but not RP‐deletion mutants. Taken together, these results clearly indicate that both CaMKK isoforms require the RP‐domain to recognize downstream kinases to interact with and phosphorylate Thr residues in their activation loops. Thus, the RP‐domain may be a promising target for specific CaMKK inhibitors.