Protein Kinase B/Akt Participates in GLUT4 Translocation by Insulin in L6 Myoblasts

L6 myoblasts stably transfected with a GLUT4 cDNA harboring an exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study the role of protein kinase B alpha (PKBalpha)/Akt1 in the insulin-induced translocation of GLUT4 to the cell surface. Surface GLUT4myc was detected by immunofluorescent labeling of the myc epitope in nonpermeabilized cells. Insulin induced a marked translocation of GLUT4myc to the plasma membrane within 20 min. This was prevented by transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (Deltap85alpha). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A constitutively active PKBalpha, created by fusion of a viral Gag protein at its N terminus (GagPKB), increased the cell surface density of GLUT4myc compared to that of neighboring nontransfected cells. A kinase-inactive, phosphorylation-deficient PKBalpha/Akt1 construct with the mutations K179A (substitution of alanine for the lysine at position 179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative inhibitor of insulin-dependent activation of cotransfected wild-type hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited the insulin-induced phosphorylation of cotransfected BAD, demonstrating inhibition of the endogenous PKB/Akt. Under the same conditions, AAA-PKB almost entirely blocked the insulin-dependent increase in surface GLUT4myc. PKBalpha with alanine substitutions T308A and S473A (AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of insulin-dependent activation of wild-type HA-PKB or GLUT4myc translocation than was AAA-PKB. Cotransfection of AAA-PKB with a fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc translocation. AAA-PKB did not prevent actin bundling (membrane ruffling), though this response was PI 3-kinase dependent. Therefore, it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling. These results outline an important role for PKBalpha/Akt1 in the stimulation of glucose transport by insulin in muscle cells in culture.     

A Role for Protein Kinase Bβ/Akt2 in Insulin-Stimulated GLUT4 Translocation in Adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKBbeta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKBalpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKBbeta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKBbeta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKBbeta in insulin-stimulated glucose transport in adipocytes.     

Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation

Death-associated protein kinase 2 (DAPK2) is a Ca²⁺/calmodulin-dependent Ser/Thr kinase that possesses tumor-suppressive functions and regulates programmed cell death, autophagy, oxidative stress, hematopoiesis, and motility. As only few binding partners of DAPK2 have been determined, the molecular mechanisms governing these biological functions are largely unknown. We report the identification of 180 potential DAPK2 interaction partners by affinity purification-coupled mass spectrometry, 12 of which are known DAPK binding proteins. A small subset of established and potential binding proteins detected in this screen was further investigated by bimolecular fluorescence complementation (BiFC) assays, a method to visualize protein interactions in living cells. These experiments revealed that α-actinin-1 and 14-3-3-β are novel DAPK2 binding partners. The interaction of DAPK2 with α-actinin-1 was localized at the plasma membrane, resulting in massive membrane blebbing and reduced cellular motility, whereas the interaction of DAPK2 with 14-3-3-β was localized to the cytoplasm, with no impact on blebbing, motility, or viability. Our results therefore suggest that DAPK2 effector functions are influenced by the protein''s subcellular localization and highlight the utility of combining mass spectrometry screening with bimolecular fluorescence complementation to identify and characterize novel protein-protein interactions.     

TRAPPC2L is a Novel, Highly Conserved TRAPP-Interacting Protein

Mutations in the trafficking protein particle complex C2 protein (TRAPPC2), a mammalian ortholog of yeast Trs20p and a component of the trafficking protein particle (TRAPP) vesicle tethering complex, have been linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT). Intriguingly, the X-linked TRAPPC2 is just one of a complement of Trs20-related genes in humans. Here we characterize TRAPPC2L, a novel, highly conserved TRAPP-interacting protein related to TRAPPC2 and the uncharacterized yeast open reading frame YEL048c . TRAPPC2L and TRAPPC2 genes are found in pairs across species and show broad and overlapping expression, suggesting they are functionally distinct, a notion supported by yeast complementation studies and biochemical characterization. RNA interference-mediated knockdown of either TRAPPC2L or TRAPPC2 in HeLa cells leads to fragmentation of the Golgi, implicating both proteins in Golgi dynamics. Gradient fractionation of cellular membranes indicates that TRAPPC2L is found with a portion of cellular TRAPP on very low-density membranes whereas the remainder of TRAPP, but not TRAPPC2L, is found associated with Golgi markers. YEL048c displays genetic interactions with TRAPP II-encoding genes and the gene product co-fractionates with and interacts with yeast TRAPP II. Taken together these results indicate that TRAPPC2L and its yeast ortholog YEL048c are novel TRAPP-interacting proteins that may modulate the function of the TRAPP II complex.     

Quantitative GTPase Affinity Purification Identifies Rho Family Protein Interaction Partners

Although Rho GTPases are essential molecular switches involved in many cellular processes, an unbiased experimental comparison of their interaction partners was not yet performed. Here, we develop quantitative GTPase affinity purification (qGAP) to systematically identify interaction partners of six Rho GTPases (Cdc42, Rac1, RhoA, RhoB, RhoC, and RhoD), depending on their nucleotide loading state. The method works with cell line or tissue-derived protein lysates in combination with SILAC-based or label-free quantification, respectively. We demonstrate that qGAP identifies known and novel binding partners that can be validated in an independent assay. Our interaction network for six Rho GTPases contains many novel binding partners, reveals highly promiscuous interaction of several effectors, and mirrors evolutionary relationships among Rho GTPases.     

蛋白质相互作用研究的新技术与新方法

目前,蛋白质相互作用已成为蛋白质组学研究的热点. 新方法的建立及对已有技术的改进标志着蛋白质相互作用研究的不断发展和完善．在技术改进方面,本文介绍了弥补酵母双杂交的蛋白定位受限等缺陷的细菌双杂交系统;根据目标蛋白特性设计和修饰TAP标签来满足复合体研究要求的串联亲和纯化技术,以及在双分子荧光互补基础上发展的动态检测多个蛋白质间瞬时、弱相互作用的多分子荧光互补技术．还综述了近两年建立的新方法：与免疫共沉淀相比,寡沉淀技术直接研究具有活性的蛋白质复合体;减量式定量免疫沉淀方法排除了蛋白质复合体中非特异性相互作用的干扰;原位操作的多表位－配基绘图法避免了样品间差异的影响,以及利用多点吸附和交联加固研究弱蛋白质相互作用的固相蛋白质组学方法.     

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.     

Functional Interaction between Ribosomal Protein L6 and RbgA during Ribosome Assembly

RbgA is an essential GTPase that participates in the assembly of the large ribosomal subunit in Bacillus subtilis and its homologs are implicated in mitochondrial and eukaryotic large subunit assembly. How RbgA functions in this process is still poorly understood. To gain insight into the function of RbgA we isolated suppressor mutations that partially restored the growth of an RbgA mutation (RbgA-F6A) that caused a severe growth defect. Analysis of these suppressors identified mutations in rplF, encoding ribosomal protein L6. The suppressor strains all accumulated a novel ribosome intermediate that migrates at 44S in sucrose gradients. All of the mutations cluster in a region of L6 that is in close contact with helix 97 of the 23S rRNA. In vitro maturation assays indicate that the L6 substitutions allow the defective RbgA-F6A protein to function more effectively in ribosome maturation. Our results suggest that RbgA functions to properly position L6 on the ribosome, prior to the incorporation of L16 and other late assembly proteins.     

BRPF1及其新型转录本BRPF2与RHOX5蛋白间的相互作用

RHOX5基因是最早发现的小鼠RHOX基因簇(reproductive homeobox on the X chromosome)成员,可特异性地在生殖系统中表达.RHOX5蛋白在胚胎发育、生殖组织的发育、精子的生成和成熟等多个环节发挥作用,但其功能的发挥途径尚不明确.在前期筛选与RHOX5蛋白相互作用的分子中初步获得一个BRPF1的新型转录本BRPF2.进一步构建pGBKT7-BRPF2质粒,酵母双杂交实验确定其与RHOX5蛋白的相互作用,GST-pull down实验确定其在体外的直接结合;PCR扩增BRPF1基因,构建pGBKT7-BRPF1和pGADT7-BRPF1质粒,酵母双杂交实验和GST-pull down实验证明RHOX5蛋白亦可以直接结合BRPF1蛋白.BRPF1及其新型转录本BRPF2与RHOX5蛋白间的相互作用证实暗示了BRPF2极有可能与BRPF1竞争性结合RHOX5蛋白,为三种蛋白功能的研究提供了新的思路.     

Analysis of Phosphorylation-dependent Protein Interactions of Adhesion and Degranulation Promoting Adaptor Protein (ADAP) Reveals Novel Interaction Partners Required for Chemokine-directed T cell Migration

Stimulation of T cells leads to distinct changes of their adhesive and migratory properties. Signal propagation from activated receptors to integrins depends on scaffolding proteins such as the adhesion and degranulation promoting adaptor protein (ADAP)¹. Here we have comprehensively investigated the phosphotyrosine interactome of ADAP in T cells and define known and novel interaction partners of functional relevance. While most phosphosites reside in unstructured regions of the protein, thereby defining classical SH2 domain interaction sites for master regulators of T cell signaling such as SLP76, Fyn-kinase, and NCK, other binding events depend on structural context. Interaction proteomics using different ADAP constructs comprising most of the known phosphotyrosine motifs as well as the structured domains confirm that a distinct set of proteins is attracted by pY571 of ADAP, including the ζ-chain-associated protein kinase of 70 kDa (ZAP70). The interaction of ADAP and ZAP70 is inducible upon stimulation either of the T cell receptor (TCR) or by chemokine. NMR spectroscopy reveals that the N-terminal SH2 domains within a ZAP70-tandem-SH2 construct is the major site of interaction with phosphorylated ADAP-hSH3^N and microscale thermophoresis (MST) indicates an intermediate binding affinity (K_d = 2.3 μm). Interestingly, although T cell receptor dependent events such as T cell/antigen presenting cell (APC) conjugate formation and adhesion are not affected by mutation of Y571, migration of T cells along a chemokine gradient is compromised. Thus, although most phospho-sites in ADAP are linked to T cell receptor related functions we have identified a unique phosphotyrosine that is solely required for chemokine induced T cell behavior.T cell migration and the establishment of productive T cell/APC interactions are regulated by the activity of integrins. In resting T cells, integrins are expressed in an inactive state that adopts a conformation with low affinity for their ligands. Members of the intercellular adhesion molecule family (ICAM 1–5) are the physiological ligands of lymphocyte function-associated antigen 1 (LFA-1, αLβ2-integrin) whereas vascular cell adhesion molecule (VCAM) and fibronectin are the ligands for the β1-integrin very late antigen 4 (VLA-4) (1, 2). Triggering of the T cell receptor (TCR) by peptide-major histocompatibility complex (MHC) or stimulation of chemokine receptors (e.g. CCR7 with CCL21 or CXCR4 with CXCL12) induces a conformational change of the integrins that increases their ligand binding (affinity regulation) and subsequently mediates clustering of integrins at the cell surface (avidity regulation). The intracellular events leading to integrin activation have collectively been termed inside-out signaling. Conversely, ligand-bound integrins transmit a signal to the T cell and thereby promote adhesion, activation, proliferation, and migration of T cells (outside-in signaling) (1, 2).In both inside-out and outside-in signaling pathways tyrosine phosphorylation of adaptor proteins, either present as transmembrane scaffolds or as transiently membrane-anchored proteins, is a crucial primary event in signal transmission to integrins.An essential functional module operating at the integrin-membrane-cytoskeleton interface contains the cytosolic adaptor protein ADAP at its core. Ablation of ADAP in mice leads to dysfunctional integrin clustering and activity, thus compromising the adhesive and migratory properties of these cells. In addition to its instantaneous effects on cellular motility, ADAP was shown to act as a regulator of NFκB p65 nuclear translocation (3), a function that might well contribute to the observed modulation of cytokine production, like interleukin-2 (4, 5). A contribution of ADAP to mast cell degranulation has been postulated (6), and its complex formation with cytoskeletal regulators during early phases of phagocytosis in macrophages has been recognized early on (7). ADAP is also critical for normal platelet adhesion (8) and mutations in the human protein have recently been suggested to form an underlying genetic cause for autosomal recessive thrombocytopenia (9).ADAP interacts with several effectors of T cell function, either constitutively or phosphorylation-dependent. The SH3 domain of SKAP55 (Src-kinase associated phosphoprotein of 55 kDa) interacts with a proline-rich sequence (PRS) stretch in ADAP (Fig. 1A) (10, 11), whereas another PRS (FPPPP) is responsible for the interaction with the actin regulator Ena/VASP-like protein (EVL) (12). Membrane binding of the ADAP-SKAP55 complex is conferred by the PH domain of SKAP55 and to a lower extend by the C-terminal hSH3 domain (hSH3^C) of ADAP (13–16). Moreover, ADAP is strongly tyrosine-phosphorylated upon TCR stimulation and thereby serves as a hub for SH2 domain-containing proteins such as SLP76, FYN and NCK (Fig. 1A) (17–21). Beside these well characterized interactions, several other SH2-domain containing binders were identified by pull-down approaches using phosphorylated peptide baits (Fig. 1A) (22, 23). Most of the so far characterized SH2-pTyr interactions in ADAP are mapped to unstructured regions. An exception is Y571, which is located in close proximity to the folded hSH3^N domain of ADAP. Phosphoproteomic profiling of activated T cells has identified Y571 as a major phosphorylation site in ADAP (18, 24–30). Our rationale for the experimental approach chosen here was that the constraints imposed by the folding of the hSH3 domains impact the choice of SH2 domains that bind to such motifs in ADAP.Open in a separate windowFig. 1.Phosphotyrosine sites of ADAP and interaction partners. A, Schematic overview of the ADAP primary structure indicating interaction partners of different phosphotyrosine sites identified by peptide pull-down approaches (22, 23). Black arrows show SH2-pTyr interaction sites. White arrows show interactions dependent on proline rich sequences (PRS). B, Fyn kinase catalyzed in vitro phosphorylation of full-length ADAP. Identification and relative quantification of phosphorylation degrees of individual tyrosine residues was obtained by mass spectrometry. Phosphorylation degrees were estimated by comparing relative MS peak intensities of the corresponding peptide/phosphopeptide pairs as described (33, 34).To meet this challenge, we employed interaction proteomics and immunoprecipitation experiments using different constructs of ADAP that comprise the major phosphotyrosine sites including the two folded hSH3 domains. In particular, Y571, a residue residing at the domain border of the hSH3^N domain, is shown to interact with proteins that are not identified by the peptide pull-down approach. We identify the ZAP70 kinase as the most robust binding partner of pY571 and show that this interaction is maintained in primary T cells. Using NMR spectroscopy we further show that the N-terminal SH2 domain of ZAP70 is responsible for binding and we confirm the direct interaction between the two proteins to be inducible upon T cell receptor or CXCR4 stimulation. Abolishing the interaction by using a Y571F mutation compromises T cell migration along a CXCL12 gradient but does not affect TCR- and CXCR4-mediated adhesion and T cell interaction with APCs. In this way, the Y571F mutant of ADAP provides the striking example of a molecular switch that selectively invokes the migratory, but not the adhesive, signaling pathways in T cells.     

PI3K/Akt在C2C12肌细胞生脂转分化中的作用

本研究的主要目的在于探明PI3K/Akt通路在肌细胞生脂转分化中的调控作用.试验培养并诱导C2C12肌细胞生脂转分化,同时使用抑制剂Wortmannin处理细胞抑制PI3K的激活,或者使用特异性siR NA转染沉默细胞内源PI3K基因的表达,观察其对肌细胞生脂转分化的影响.结果表明,随着C2C12细胞的生脂转分化,PI3K蛋白(P55亚基和P85亚基)和其下游效应分子Akt的磷酸化水平,在转分化前期提高而在转分化后期明显降低.使用Wortmannin处理细胞能够有效抑制PI3K/Akt激活,这导致C2C12细胞的生脂转分化明显受到抑制,细胞内脂肪生成量显著降低,生脂基因PPARγ、C/EBPα、FABP4和FATP1的表达水平均显著下调.使用特异性siR NA转染细胞显著下调PI3K基因表达水平和蛋白质含量,同样明显抑制了C2C12细胞的生脂转分化.此外,在转分化过程中抑制PI3K/Akt的活性和表达还激活了Caspase-3并导致细胞凋亡.综合上述结果可以确认PI3K/Akt的正常表达和激活是肌细胞生脂转分化必不可少的.     

Novel Endogenous Protein Kinase C Substrate Proteins, Neutrinins, in Brain Synaptic Membranes of Maturing Rat

Abstract: A new family of membrane phosphoproteins designated as P9, P12, P15, P16, and P20 with corresponding apparent molecular weights of 9K, 12K, 15K, 16K, and 20K was characterized from rat brain by using in vitro exogenous or endogenous phosphorylation and autoradiography. As the phosphorylation was selectively inhibited by the protein kinase C (PKC) inhibitor PKC_19–31 or Ca²⁺-chelating reagents and again stimulated by the PKC activator phorbol 12,13-dibutyrate, these proteins are thought to be the natural PKC substrates. Because P12, P15, P16, and P20 were neutral proteins (pl 7.0) and specifically distributed in neuronal membranes, the new family of membrane-associated PKC substrate proteins was referred to as neutrinins. Neutrinins were widely distributed in rat brain, being especially plentiful in the spinal cord, medulla oblongata, cerebellum, and midbrain, relatively scanty in the cerebral cortex, but lacking in cytosol of brain areas and cell membrane preparations of peripheral tissues. The expression of the developmental changes of neutrinins has been monitored by the in vitro exogenous phosphorylation approach, i.e., adding purified PKC to a deactivated synaptosomal plasma membrane system. Levels of all the neutrinin proteins in rat cerebral cortex, as represented by P12, P15, and P16, showed an ontogenetic increase from the early postnatal days to the adult. This appears to be correlated with the commencement of synaptogenesis.     

Retinoic Acid and Its Geometrical Isomers Block Both Growth and Fusion of L6 Myoblasts by Modulating the Expression of Protein Kinase A

All-trans retinoic acid (RA) and its geometrical isomers, such as 9-cis RA, 13-cis RA, and 9,13-di-cis RA, strongly inhibited both growth and fusion of L6 myoblasts. However, illumination of white light diminished their inhibitory activity on membrane fusion with little effect on cell growth. During myogenic differentiation, the intracellular level of cAMP decreased whereas the total activity of protein kinase A as well as the protein level of its regulatory subunit I (RI) and catalytic subunit (C) increased. RAs raised the intracellular level of cAMP by over 3-fold, but decreased the total activity of protein kinase A. Like RAs, dibutyryl-cAMP inhibited myoblast fusion and reduced the expression of both RI and C subunits. These results suggest that RAs negatively modulate the differentiation of L6 myoblasts by increasing the intracellular level of cAMP, which may in turn down-regulate the expression of protein kinase A and hence its activity.     

Identification of a Novel Protein Interaction Motif in the Regulatory Subunit of Casein Kinase 2

Casein kinase 2 (CK2) regulates multiple cellular processes and can promote oncogenesis. Interactions with the CK2β regulatory subunit of the enzyme target its catalytic subunit (CK2α or CK2α′) to specific substrates; however, little is known about the mechanisms by which these interactions occur. We previously showed that by binding CK2β, the Epstein-Barr virus (EBV) EBNA1 protein recruits CK2 to promyelocytic leukemia (PML) nuclear bodies, where increased CK2-mediated phosphorylation of PML proteins triggers their degradation. Here we have identified a KSSR motif near the dimerization interface of CK2β as forming part of a protein interaction pocket that mediates interaction with EBNA1. We show that the EBNA1-CK2β interaction is primed by phosphorylation of EBNA1 on S393 (within a polyserine region). This phosphoserine is critical for EBNA1-induced PML degradation but does not affect EBNA1 functions in EBV replication or segregation. Using comparative proteomics of wild-type (WT) and KSSR mutant CK2β, we identified an uncharacterized cellular protein, C18orf25/ARKL1, that also binds CK2β through the KSSR motif and show that this involves a polyserine sequence resembling the CK2β binding sequence in EBNA1. Therefore, we have identified a new mechanism of CK2 interaction used by viral and cellular proteins.     

Effect of Isopentenyladenine,a Cytokinin,on Proliferation and Protein Synthesis in Cultured Myoblasts

The effects of cytokinins, a class of plant hormones, on cell proliferation and protein synthesis were studied in rat-derived L₆ myoblasts cultured in a serum-free medium. Of the three cytokinins tested, isopentenyladenine, zeatin and ribosylzeatin, isopentenyladenine (5 μm) most stimulated the growth and DNA synthesis of the myoblasts, and it dose-dependently (0 ~ 10μm) enhanced the proliferation and DNA synthesis of the cells. Isopentenyladenine (5 and 10 μm) increased protein synthesis to twice that of control (0μm). These results suggest that isopentenyladenine, a trace component in plant food and a plant hormone, can affect the metabolism of an animal cell line of myoblasts.