Macroautophagy/autophagy plays key roles in development, oncogenesis, and cardiovascular and metabolic diseases. Autophagy-specific class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) is essential for autophagosome formation. However, the regulation of this complex formation requires further investigation. Here, we discovered that STYK1 (serine/threonine/tyrosine kinase 1), a member of the receptor tyrosine kinases (RTKs) family, is a new upstream regulator of autophagy. We discovered that STYK1 facilitated autophagosome formation in human cells and zebrafish, which was characterized by elevated LC3-II and lowered SQSTM1/p62 levels and increased puncta formation by several marker proteins, such as ATG14, WIPI1, and ZFYVE1. Moreover, we observed that STYK1 directly binds to the PtdIns3K-C1 complex as a homodimer. The binding with this complex was promoted by Tyr191 phosphorylation, by means of which the kinase activity of STYK1 was elevated. We also demonstrated that STYK1 elevated the serine phosphorylation of BECN1, thereby decreasing the interaction between BECN1 and BCL2. Furthermore, we found that STYK1 preferentially facilitated the assembly of the PtdIns3K-C1 complex and was required for PtdIns3K-C1 complex kinase activity. Taken together, our findings provide new insights into autophagy induction and reveal evidence of novel crosstalk between the components of RTK signaling and autophagy.

Abbreviations: AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: adenosine 5‘-monophosphate (AMP)-activated protein kinase; ATG: autophagy related; ATP: adenosine triphosphate; BCL2: BCL2 apoptosis regulator; BECN1: beclin 1; Bre A: brefeldin A; Co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DAPI: 4′,6-diamidino-2-phenylindole; EBSS: Earle’s balanced salt solution; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GSEA: gene set enrichment analysis; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAPK8/JNK1: mitogen-activated protein kinase 8; mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; MTT: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; qRT-PCR: quantitative reverse transcription PCR; RACK1: receptor for activated C kinase 1; RUBCN: rubicon autophagy regulator; siRNA: small interfering RNA; SQSTM1: sequestosome 1; STYK1/NOK: serine/threonine/tyrosine kinase 1; TCGA: The Cancer Genome Atlas; Ub: ubiquitin; ULK1: unc-51 like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; WIPI1: WD repeat domain, phosphoinositide interacting 1; ZFYVE1: zinc finger FYVE-type containing 1

巨自噬/自噬在发育、肿瘤发生以及心血管和代谢疾病中起着关键作用。自噬特异性 III 类磷脂酰肌醇 3-激酶复合物 I（PtdIns3K-C1）对于自噬体的形成至关重要。然而，这种复杂形成的调控需要进一步研究。在这里，我们发现 STYK1（丝氨酸/苏氨酸/酪氨酸激酶 1）是受体酪氨酸激酶 (RTK) 家族的成员，是一种新的自噬上游调节剂。我们发现 STYK1 促进了人类细胞和斑马鱼中自噬体的形成，其特征是 LC3-II 升高，SQSTM1/p62 水平降低，并增加了几种标记蛋白（如 ATG14、WIPI1 和 ZFYVE1）的斑点形成。此外，我们观察到 STYK1 作为同型二聚体直接与 PtdIns3K-C1 复合物结合。Tyr191 磷酸化促进了与该复合物的结合，从而提高了 STYK1 的激酶活性。我们还证明 STYK1 提高了 BECN1 的丝氨酸磷酸化，从而降低了 BECN1 和 BCL2 之间的相互作用。此外，我们发现 STYK1 优先促进 PtdIns3K-C1 复合物的组装，并且是 PtdIns3K-C1 复合物激酶活性所必需的。总之，我们的研究结果提供了对自噬诱导的新见解，并揭示了 RTK 信号和自噬成分之间新串扰的证据。我们发现 STYK1 优先促进 PtdIns3K-C1 复合物的组装，并且是 PtdIns3K-C1 复合物激酶活性所必需的。总之，我们的研究结果提供了对自噬诱导的新见解，并揭示了 RTK 信号和自噬成分之间新串扰的证据。我们发现 STYK1 优先促进 PtdIns3K-C1 复合物的组装，并且是 PtdIns3K-C1 复合物激酶活性所必需的。总之，我们的研究结果提供了对自噬诱导的新见解，并揭示了 RTK 信号和自噬成分之间新串扰的证据。

缩写：AICAR：5-氨基咪唑-4-甲酰胺核糖核苷酸；AMPK：腺苷 5'-单磷酸 (AMP) 活化蛋白激酶；ATG：自噬相关；ATP：三磷酸腺苷；BCL2：BCL2 凋亡调节剂；BECN1: beclin 1; Bre A：brefeldin A；Co-IP：共免疫沉淀；CRISPR：成簇的规则间隔短回文重复；DAPI：4',6-二脒基-2-苯基吲哚；EBSS：厄尔平衡盐溶液；GAPDH：3-磷酸甘油醛脱氢酶；GFP：绿色荧光蛋白；GSEA：基因集富集分析；MAP1LC3/LC3，微管相关蛋白1轻链3；MAPK8/JNK1：丝裂原活化蛋白激酶 8；mRFP：单体红色荧光蛋白；MTOR：雷帕霉素激酶的机制靶点；MTT: 3-(4,5-二甲基噻唑-2-基)-2, 5-二苯基溴化四唑；PIK3C3：磷脂酰肌醇 3-激酶催化亚基 3 型；PIK3R4：磷酸肌醇-3-激酶调节亚基 4；qRT-PCR：定量逆转录PCR；RACK1：活化 C 激酶 1 的受体；RUBCN：rubicon自噬调节剂；siRNA：小干扰RNA；SQSTM1：螯合体 1；STYK1/NOK：丝氨酸/苏氨酸/酪氨酸激酶 1；TCGA：癌症基因组图谱；Ub：泛素；ULK1：unc-51 类似自噬激活激酶 1；UVRAG：相关的抗紫外线辐射；WIPI1：WD 重复结构域，磷酸肌醇相互作用 1；ZFYVE1：锌指FYVE型含1 unc-51 像自噬激活激酶 1；UVRAG：相关的抗紫外线辐射；WIPI1：WD 重复结构域，磷酸肌醇相互作用 1；ZFYVE1：锌指FYVE型含1 unc-51 像自噬激活激酶 1；UVRAG：相关的抗紫外线辐射；WIPI1：WD 重复结构域，磷酸肌醇相互作用 1；ZFYVE1：锌指FYVE型含1