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Ubl4a Is Required for Insulin-Induced Akt Plasma Membrane Translocation Through Promotion of Arp2/3-Dependent Actin Branching

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Ubl4a Is Required for Insulin-Induced Akt Plasma Membrane Translocation Through Promotion of Arp2/3-Dependent Actin Branching Ubl4A is required for insulin-induced Akt plasma membrane translocation through promotion of Arp2/3-dependent actin branching Yu Zhaoa, Yuting Lina, Honghong Zhanga, Adriana Mañasa, Wenwen Tangb, Yuzhu Zhangc, Dianqing Wub, Anning Linc, and Jialing Xianga,1 aDepartment of Biology, Illinois Institute of Technology, Chicago, IL 60616; bDepartment of Pharmacology, Yale University School of Medicine, New Haven, CT 06520; and cBen May Department for Cancer Research, University of Chicago, Chicago, IL 60637 Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 1, 2015 (received for review May 6, 2015) The serine-threonine kinase Akt is a key regulator of cell prolifer- Results ation and survival, glucose metabolism, cell mobility, and tumor- Ubl4A-Deficient Mice Display Increased Neonatal Mortality and a igenesis. Activation of Akt by extracellular stimuli such as insulin Defect in Liver Glycogen Synthesis. To better understand the bi- centers on the interaction of Akt with PIP3 on the plasma mem- ological functions of Ubl4A, we generated Ubl4A-deficient mice brane, where it is subsequently phosphorylated and activated (Fig. S1). Ubl4A knockout (KO) mice were viable but displayed by upstream protein kinases. However, it is not known how Akt increased neonatal mortality (occurring mostly within 24 h after is recruited to the plasma membrane upon stimulation. Here we A report that ubiquitin-like protein 4A (Ubl4A) plays a crucial role in birth) compared with their wild type (WT) littermates (Fig. 1 ). insulin-induced Akt plasma membrane translocation. Ubl4A knock- We noticed that some Ubl4A KO pups displayed signs of cyanosis A Inset out newborn mice have defective Akt-dependent glycogen syn- before death (Fig. 1 , ). The surviving Ubl4A KO newborns, thesis and increased neonatal mortality. Loss of Ubl4A results in however, appeared to be normal despite modestly low body weight the impairment of insulin-induced Akt translocation to the plasma (Fig. 1B). We wondered that increased mortality of Ubl4A KO membrane and activation. Akt binds actin-filaments and colocal- neonates might be the result of defective glucose homeostasis, as izes with actin-related protein 2 and 3 (Arp2/3) complex in the glycogenolysis and mobilization of glycogen storage from liver are membrane ruffles and lamellipodia. Ubl4A directly interacts with vital for the newborns to combat starvation-induced hypoglycemia Arp2/3 to accelerate actin branching and networking, allowing Akt (15). Indeed, glycogen staining analysis revealed that WT neonatal to be in close proximity to the plasma membrane for activation livers had typical abundant accumulation of glycogen ∼5 h after upon insulin stimulation. Our finding reveals a new mechanism birth (Fig. 1 C and D), which was quickly consumed 10 h after by which Akt is recruited to the plasma membrane for activation, birth (Fig. S2). In contrast, glycogen synthesis was significantly thereby providing a missing link in Akt signaling. lower in Ubl4A KO livers than in WT livers during the same period (Fig. 1 C and D). Consistently, phosphorylation of liver Ubl4A | Akt translocation | Arp2/3 complex | actin branching | insulin glycogen synthase (GS), which is an effector in the Akt-GS kinase 3β (GSK3β) pathway (16), was increased in the primary KO he- he serine-threonine kinase Akt (also known as protein kinase patocytes compared with the WT counterparts (Fig. 1E), sug- B) is a key regulator of cell proliferation and survival, glucose T gesting the activity of GS was decreased. Furthermore, the metabolism, cell mobility, and tumorigenesis, and its dysregulation β has been implicated in human diseases (1–3). Akt is activated by a inhibitory phosphorylation of GSK3 , which is the upstream ki- variety of extracellular stimuli including insulin (4). Upon stimula- nase of GS, was decreased in the Ubl4A KO hepatocyte cells (Fig. tion, Akt is recruited to the plasma membrane by phosphatidyli- 1E). Thus, the defect in liver glycogen synthesis may contribute to, nositol (3-5)-triphosphate (PIP3), which interacts directly with the at least in part, the increased neonatal mortality and growth Akt pleckstrin homology (PH) domain, and is subsequently phos- retardation in Ubl4A-deficient mice. phorylated at Thr308 and Ser473, and thus activated, by protein kinases such as phosphoinositide-dependent kinase-1 (PDK1) and Significance mammalian target of rapamycin (mTOR) complex 2 (mTORC2) – (5 8). It is, however, not known how Akt translocates to the plasma The signal-induced Akt membrane recruitment is a crucial step for membrane in response to extracellular signals such as insulin. its activation, but the underlying mechanism is incompletely un- Ubl4A (also known as Gdx in mammalian or Get5 in yeast) is derstood. We show that ubiquitin-like protein 4A (Ubl4A) is re- a small ubiquitin-like protein encoded by an X-linked house- quired for insulin-induced Akt membrane translocation through keeping gene (9). Although Ubl4A contains an ubiquitin-like direct promotion of actin-related protein 2 and 3 (Arp2/3)- domain at its N terminus, it lacks ubiquitination activity (10). In dependent actin branching, thereby ensuring glycogen synthesis yeast, Ubl4A homolog Get5 forms a complex with Get4 to assist for neonatal survival. As a novel Arp2/3-binding protein, Ubl4A Get3 for recruitment of nascent synthesized tail-anchored (TA) may play important roles in the translocation of other actin-bind- proteins and their delivery to the endoplasmic reticulum (11, 12). ing molecules controlled by a similar mechanism, as well as a broad Ubl4A and other mammalian Get homologs are also involved in range of cellular functions related to the actin branching network. the recruitment and delivery of TA protein into the endoplasmic reticulum membrane (11, 13). Furthermore, Ubl4A can also Author contributions: Y. Zhao, D.W., and J.X. designed research; Y. Zhao, Y.L., H.Z., A.M., suppress tumorigenesis through regulation of signal transducer W.T., and Y. Zhang performed research; Y. Zhao, Y.L., D.W., A.L., and J.X. analyzed and activation of transcription 3 (13) and is involved in DNA data; and Y. Zhao, D.W., A.L., and J.X. wrote the paper. damage-mediated cell death (14). Here we report that Ubl4A is The authors declare no conflict of interest. required for insulin-induced Akt plasma membrane translocation This article is a PNAS Direct Submission. and activation through direct promotion of Arp2/3-dependent 1To whom correspondence should be addressed. Email: [email protected]. actin branching, thereby playing a critical role in regulation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. glycogen synthesis for neonatal survival. 1073/pnas.1508647112/-/DCSupplemental. 9644–9649 | PNAS | August 4, 2015 | vol. 112 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1508647112 Downloaded by guest on September 28, 2021 by the significant reduction of phosphorylated endogenous Akt on the plasma membrane in Ubl4A KO cells, as analyzed using confocal microscope imaging (Fig. 2 E and F). Thus, loss of Ubl4A mainly affects Akt plasma membrane translocation. Regulation of Akt Membrane Translocation by Ubl4A Depends on the Actin Network and the Arp2/3 Complex. To determine how Ubl4A regulates the translocation of Akt from the cytoplasm to the plasma membrane, a step about which very little is known, WT and KO primary fibroblasts were transfected with expression vector-encoding GFP-tagged Akt-PH domain (Akt-PH-GFP). Time-lapse microscopy revealed that upon insulin stimulation, Akt-PH-GFP proteins formed actin filament-like structures and appeared to be more readily recruited to the plasma membrane- ruffling regions in WT than in Ubl4A-deficient fibroblasts (Fig. 3A and Movies S1 and S2). Because Akt-PH-GFP seemed to be more extensively recruited to the structures similar to actin filaments and subsequently accumulated in membrane-ruffling regions (Fig. 3A), we examined the colocalization of F-actin and Akt-PH-GFP upon insulin stimulation. Confocal immunofluo- rescence microscopy revealed that Akt-PH-GFP proteins indeed Fig. 1. Ubl4A-deficient mice display increased neonatal mortality and a defect in liver glycogen synthesis. (A) The survival rate of neonates within 24 h after birth. (Inset) Representative WT and KO littermates ∼5 h after birth. (B) The body weight of 3-wk-old mice; data shown are mean ± SEM. WT, n = 37; KO, n = 50. **P < 0.01. (C) Liver glycogen PAS staining of the neonates ∼5hafterbirth.(Scalebar,100μm.) (D) Quantitative analysis of liver PAS staining by matrix score. The average percentage of PAS positive area is scored as 0 (0%), 1 (25%), 3 (75%), or 4 (100%). Error bars present SD. n = 9. ***P < 0.001. (E) Immunoblotting analysis of phosphorylated and total GSK3β and GS of primary hepatocytes. Loss of Ubl4A Results in Impaired Akt Plasma Membrane Translocation and Activation. The defect in glycogen synthesis in Ubl4A KO mice led us to investigate activation of the GSK3β upstream apical kinase Akt. Insulin-induced Akt phosphorylation at Ser473 and Thr308, both of which are required for Akt activation on the plasma membrane (17), was significantly decreased in Ubl4A KO primary hepatocytes and mouse embryonic fibroblasts (MEFs) compared with their WT counterparts (Fig. 2A). As expected, phosphorylation of S6K, an Akt substrate, was impaired in Ubl4A-deficient MEFs (Fig. S3A). In contrast, Akt phosphorylation at Thr450, which is involved in stabilization of newly synthesized Akt in the cytoplasm (18), was not affected (Fig. S3B). The impaired activation of Akt CELL BIOLOGY could be a result of defects either in Akt translocation from the cytoplasm to the plasma membrane or in its upstream components such as PIP3, mTOR, or PDK1 on the plasma membrane. The latter is unlikely. There was no significant difference in production of PIP3 between insulin-treated WT and KO fibroblasts (Fig. 2B C and Fig. S3 ). Consistently, the addition of exogenous PIP3 to the Fig.
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