Serine/threonine kinase 16 and MAL2 regulate constitutive secretion of soluble cargo in hepatic cells.
Serine/threonine kinase 16 and MAL2 regulate constitutive secretion of soluble cargo in hepatic cells.
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DOI：
10.1042/bj20140468
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发表时间：
2014-10-15
期刊：
影响因子：
--
通讯作者：
Tuma PL
中科院分区：
文献类型：
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作者：
In JG;Striz AC;Bernad A;Tuma PL
MAL2(髓鞘和淋巴细胞蛋白2)被认为在肝脏顶端跨细胞途径中至少调节两个步骤。由于每一步囊泡的萌发和传递都是由复杂的机制驱动的，我们预测MAL2参与了几个具有多个结合伙伴的大型蛋白质复合体。为了确定新的MAL2相互作用因子，我们进行了裂解泛素酵母双杂交实验，确定STK16(丝氨酸/苏氨酸激酶16)是一个可能的相互作用因子，我们从形态和生化上证实了这一点。由于STK16是一种高尔基体相关的结构性活性蛋白，参与调节分泌，而且肝细胞具有巨大的结构性分泌能力，我们测试了MAL2和STK16是否在分泌中起作用。显性负性激酶死亡的STK16突变体(E202a)的表达或MAL2的敲除会损害分泌，这与白蛋白和结合珠蛋白的表达减少有关。通过19℃温度阻断和溶酶体脱酸，我们确定E202a的表达或MAL2基因敲除不会干扰白蛋白的合成或加工，但会导致白蛋白溶酶体的降解。我们的结论是，MAL2和结构性活性的STK16在极化肝细胞的TGN(Trans-Golgi Network)处起到将分泌性可溶性货物分类到结构性分泌途径中的作用。
MAL2 (myelin and lymphocyte protein 2) is thought to regulate at least two steps in the hepatic apical transcytotic pathway. As vesicle budding and delivery at each step are driven by complex machineries, we predicted that MAL2 participates in several large protein complexes with multiple binding partners. To identify novel MAL2 interactors, we performed split-ubiquitin yeast two-hybrid assays and identified STK16 (serine/threonine kinase 16) as a putative interactor which we verified morphologically and biochemically. As STK16 is a Golgi-associated constitutively active kinase implicated in regulating secretion and because of the massive constitutive secretory capacity of hepatic cells, we tested whether MAL2 and STK16 function in secretion. Expression of a dominant-negative kinase-dead STK16 mutant (E202A) or knockdown of MAL2 impaired secretion that correlated with decreased expression of albumin and haptoglobin. By using 19 °C temperature blocks and lysosome deacidification, we determined that E202A expression or MAL2 knockdown did not interfere with albumin synthesis or processing, but led to albumin lysosomal degradation. We conclude that MAL2 and the constitutively active STK16 function to sort secretory soluble cargo into the constitutive secretory pathway at the TGN (trans-Golgi network) in polarized hepatocytes.