Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities.
Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities.
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DOI：
10.1016/j.jmb.2011.07.033
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发表时间：
2011-09-09
影响因子：
5.6
通讯作者：
West AB
中科院分区：
文献类型：
--
作者：
Webber PJ;Smith AD;Sen S;Renfrow MB;Mobley JA;West AB
LRRK2 蛋白同时具有 GTP 酶和激酶活性，任一酶结构域的突变都可能导致迟发性帕金森病 (PD)。 GTPase 结构域中的核苷酸结合可能是激酶活性所必需的，并且 GTPase 结构域中的残基是自磷酸化的潜在位点，表明内在调节的复杂机制。为了进一步确定 LRRK2 自磷酸化的影响，我们应用了一种最适合检测蛋白质磷酸化、电子转移解离 (ETD) 的技术，并鉴定了仅在 GTPase 结构域中的核苷酸结合口袋附近的自磷酸化事件。 PD 相关突变改变激酶活性，但不改变自磷酸化位点特异性或强大的体外底物髓磷脂碱性蛋白中的磷酸化位点。 GTPase 结构域中的氨基酸取代对激酶活性有很大影响，因为插入 GTPase 相关的 R1441C 致病性突变以及 G2019S 激酶结构域突变会导致活性成倍增加（约 7 倍）。通过突变为丙氨酸残基来去除保守的自磷酸化位点 (T1503)，导致 GTP 结合和激酶活性大大降低。虽然自磷酸化可能有助于增强激酶活性，但我们发现寡聚化和活性二聚体种类的丢失以不依赖于 ATP 和自磷酸化的方式发生。 LRRK2 自磷酸化位点在体外总体上受到强有力的保护，免于去磷酸化，这表明对体内活性的严格控制。我们开发了针对 pT1503 的高度特异性抗体，但未能检测到来自转基因小鼠和细胞系的蛋白质中的内源性自磷酸化。 LRRK2 体内活性不太可能是组成型的，而是针对特定反应进行细化的。
The LRRK2 protein has both GTPase and kinase activities and mutation in either enzymatic domain can cause late-onset Parkinson’s disease (PD). Nucleotide binding in the GTPase domain may be required for kinase activity and residues in the GTPase domain are potential sites for autophosphorylation, suggesting a complex mechanism of intrinsic regulation. To further define the effects of LRRK2 autophosphorylation, we applied a technique optimal for detection of protein phosphorylation, electron transfer dissociation (ETD), and identified autophosphorylation events exclusively nearby the nucleotide binding pocket in the GTPase domain. PD-linked mutations alter kinase activity but did not alter autophosphorylation site specificity or sites of phosphorylation in a robust in vitro substrate myelin basic protein. Amino-acid substitutions in the GTPase domain have large effects on kinase activity, as insertion of the GTPase-associated R1441C pathogenic mutation together with the G2019S kinase-domain mutation resulted in a multiplicative increase (~7-fold) in activity. Removal of a conserved autophosphorylation site (T1503) by mutation to an alanine residue resulted in greatly decreased GTP-binding and kinase activity. While autophosphorylation likely serves to potentiate kinase activity, we find that oligomerization and loss of the active dimer species occurs in an ATP and autophosphorylation independent manner. LRRK2 autophosphorylation sites are overall robustly protected from dephosphorylation in vitro, suggesting tight control over activity in vivo. We developed highly specific antibodies targeting pT1503 but failed to detect endogenous autophosphorylation in protein derived from transgenic mice and cell lines. LRRK2 activity in vivo is unlikely to be constitutive but rather refined to specific responses.
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