Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif
Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif
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DOI：
10.1073/pnas.082235699
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发表时间：
2002-06-11
影响因子：
11.1
通讯作者：
Kornfeld, SA
中科院分区：
文献类型：
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作者：
Doray, B;Bruns, K;Kornfeld, SA
GGAs（高尔基定位蛋白，γ适应蛋白同源结构域，arf结合蛋白）是一个涉及蛋白质从高尔基体转运到内体/溶酶体的蛋白家族。这些蛋白具有模块化结构，具有n端VHS （VPS-27， Hrs和STAM）结构域，随后是GAT （GGA和TOM1）结构域，连接铰链段和c端GAE （γ -adaptin ear）结构域。分离的VHS结构域已被证明与甘露糖6-磷酸受体细胞质尾部的酸性簇(AC)-二亮氨酸基序特异性结合。在这里，我们报道了全长细胞质GGA1和GGA3，而不是GGA2，由于自身抑制，与阳离子非依赖性甘露糖6-磷酸受体结合非常差。这种抑制是由于铰链段中的AC-LL序列与VHS结构域的配体结合位点结合引起的。这种抑制作用取决于位于AC-LL基序上游三个残基的丝氨酸的磷酸化。丝氨酸在体外实验中被酪蛋白激酶2磷酸化。将GGA1抑制序列替换到GGA2中缺乏AC-LL基序的类似位置，导致后者蛋白的自抑制。这些数据表明GGA1和GGA3的活性受到磷酸化、去磷酸化循环的调节。
The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes/lysosomes. These proteins have modular structures with an N-terminal VHS (VPS-27, Hrs, and STAM) domain followed by a GAT (GGA and TOM1) domain, a connecting hinge segment, and a C-terminal GAE (gamma-adaptin ear) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in in vitro assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation, dephosphorylation.