Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.
Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms.
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DOI：
10.1042/bcj20200937
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发表时间：
2021-02-12
期刊：
影响因子：
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通讯作者：
Alessi DR
中科院分区：
文献类型：
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作者：
Malik AU;Karapetsas A;Nirujogi RS;Mathea S;Chatterjee D;Pal P;Lis P;Taylor M;Purlyte E;Gourlay R;Dorward M;Weidlich S;Toth R;Polinski NK;Knapp S;Tonelli F;Alessi DR
增强激酶活性的LRRK 2常染色体显性突变导致帕金森病。LRRK 2磷酸化Rab GTP酶的一个子集，包括其效应子结合基序内的Rab 8A和Rab 10。在这里，我们探讨是否LRRK 1，一个较少研究的同源LRRK 2调节生长因子受体贩运和破骨细胞生物学也可能磷酸化Rab蛋白。使用质谱，我们发现在LRRK 1敲除细胞中，Rab 7A在Ser 72的磷酸化受到最大影响。该残基位于Rab 8A和Rab 10上LRRK 2靶向的等同位点。因此，重组LRRK 1在Ser 72处有效磷酸化Rab 7A，但不磷酸化Rab 8A或Rab 10。采用一种新的磷酸化特异性抗体，我们发现，佛波醇酯刺激小鼠胚胎成纤维细胞显着增强Rab 7A在Ser 72通过LRRK 1的磷酸化。我们鉴定了两个LRRK 1突变（K746 G和I1412 T），相当于LRRK 2 R1441 G和I2020 T帕金森氏突变，它们增强了LRRK 1介导的Rab 7A磷酸化。我们证明了LRRK 2的两个调节子Rab 29和VPS 35 [D 620 N]不影响LRRK 1。广泛使用的LRRK 2抑制剂不抑制LRRK 1，但我们确定了一种名为GZD-824的混杂抑制剂，可抑制LRRK 1和LRRK 2。PPM 1H Rab磷酸酶在过表达时使Rab 7A去磷酸化。最后，Rab 7A与其效应RILP的相互作用不受LRRK 1磷酸化的影响，我们观察到TBK 1或PINK 1通路的最大刺激不会提高Rab 7A磷酸化。总之，这些发现加强了这样的想法，即LRRK酶已经进化为具有不同底物特异性的Rab生物学的主要调节因子。
Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.