The Structural Differences between a Glycoprotein Specific F-Box Protein Fbs1 and Its Homologous Protein FBG3

The Skp1-Cul1-F-box protein (SCF) complex catalyzes protein ubiquitination in diverse cellular processes and is one of the best-characterized ubiquitin ligases. F-box proteins determine the substrate specificities of SCF ubiquitin ligases. Among these, Fbs1/FBG1/FBXO2, Fbs2/FBG2/FBXO6, and Fbs3/FBG5/FBXO27 recognize the N-glycans of glycoproteins, whereas FBG3/FBXO44 has no sugar-binding activity, despite the high sequence homology and conservation of the residues necessary for oligosaccharide binding between Fbs1–3 and FBG3. Here we determined the crystal structure of the Skp1–FBG3 complex at a resolution of 2.6 Å. The substrate-binding domain of FBG3 is composed of a 10-stranded antiparallel β-sandwich with three helices. Although the overall structure of FBG3 is similar to that of Fbs1, the residues that form the Fbs1 carbohydrate-binding pocket failed to be superposed with the corresponding residues of FBG3. Structure-based mutational analysis shows that distinct hydrogen bond networks of four FBG3 loops, i.e., β2-β3, β5-β6, β7-β8, and β9-β10, prevent the formation of the carbohydrate-binding pocket shown in Fbs1.     

The Wnt Frizzled Receptor MOM-5 Regulates the UNC-5 Netrin Receptor through Small GTPase-Dependent Signaling to Determine the Polarity of Migrating Cells

Wnt and Netrin signaling regulate diverse essential functions. Using a genetic approach combined with temporal gene expression analysis, we found a regulatory link between the Wnt receptor MOM-5/Frizzled and the UNC-6/Netrin receptor UNC-5. These two receptors play key roles in guiding cell and axon migrations, including the migration of the C. elegans Distal Tip Cells (DTCs). DTCs migrate post-embryonically in three sequential phases: in the first phase along the Antero-Posterior (A/P) axis, in the second, along the Dorso-Ventral (D/V) axis, and in the third, along the A/P axis. Loss of MOM-5/Frizzled function causes third phase A/P polarity reversals of the migrating DTCs. We show that an over-expression of UNC-5 causes similar DTC A/P polarity reversals and that unc-5 deficits markedly suppress the A/P polarity reversals caused by mutations in mom-5/frizzled. This implicates MOM-5/Frizzled as a negative regulator of unc-5. We provide further evidence that small GTPases mediate MOM-5’s regulation of unc-5 such that one outcome of impaired function of small GTPases like CED-10/Rac and MIG-2/RhoG is an increase in unc-5 function. The work presented here demonstrates the existence of cross talk between components of the Netrin and Wnt signaling pathways and provides further insights into the way guidance signaling mechanisms are integrated to orchestrate directed cell migration.     

Phosphorylation of the Cool-1/β-Pix Protein Serves as a Regulatory Signal for the Migration and Invasive Activity of Src-transformed Cells

Previously we showed that Cool-1 (Cloned out of library-1)/β-Pix (Pak-interactive exchange factor) is phosphorylated at a specific tyrosine residue (Tyr-442) in a Src-dependent manner and serves as a dual function guanine nucleotide exchange factor (GEF)/signaling-effector for Cdc42 that is essential for transformation by Src. Here, we show that knocking-down Cool-1 or overexpressing a Cool-1 mutant that contains substitutions within its Dbl homology domain and is defective for GEF activity, inhibits Src-promoted cell migration. Similarly, the expression of a Cool-1 mutant containing a tyrosine to phenylalanine substitution at position 442, making it incapable of being phosphorylated in response to serum, epidermal growth factor (EGF), or Src, also causes a significant inhibition of the migration and invasive activity of cells expressing oncogenic Src. We further demonstrate that the phosphorylation of Cool-1 at Tyr-442 weakens its ability to bind to one of its primary interaction-partners, Cat-1 (Cool-associated tyrosine phosphosubstrate-1)/Git-1 (G protein-coupled receptor kinase-interactor-1), thus making Cat more accessible for binding to paxillin. This enables cells to alternate between states where they contain large numbers of focal complexes (i.e. conditions favoring Cool-1-Cat interactions) versus reduced numbers of focal complexes (conditions favoring Cat-paxillin interactions). Overall, these findings show that the phosphorylation-dephosphorylation cycle of Cool-1 at Tyr-442 can serve as a key regulatory signal for focal complex assembly-disassembly, and consequently, for the migration and invasive activity of Src-transformed cells.     

Amer2 Protein Interacts with EB1 Protein and Adenomatous Polyposis Coli (APC) and Controls Microtubule Stability and Cell Migration

EB1 is key factor in the organization of the microtubule cytoskeleton by binding to the plus-ends of microtubules and serving as a platform for a number of interacting proteins (termed +TIPs) that control microtubule dynamics. Together with its direct binding partner adenomatous polyposis coli (APC), EB1 can stabilize microtubules. Here, we show that Amer2 (APC membrane recruitment 2), a previously identified membrane-associated APC-binding protein, is a direct interaction partner of EB1 and acts as regulator of microtubule stability together with EB1. Amer2 binds to EB1 via specific (S/T)xIP motifs and recruits it to the plasma membrane. Coexpression of Amer2 and EB1 generates stabilized microtubules at the plasma membrane, whereas knockdown of Amer2 leads to destabilization of microtubules. Knockdown of Amer2, APC, or EB1 reduces cell migration, and morpholino-mediated down-regulation of Xenopus Amer2 blocks convergent extension cell movements, suggesting that the Amer2-EB1-APC complex regulates cell migration by altering microtubule stability.     

SRC-1, a non-receptor type of protein tyrosine kinase, controls the direction of cell and growth cone migration in C. elegans

Src family tyrosine kinase (SFK) has been implicated in the regulation of cell adhesion and migration during animal development. We show that SRC-1, an ortholog of SFK, plays an essential role in directing cell migration in Caenorhabditis elegans. The mutation in the src-1 gene results in defective distal tip cell (DTC)-directed gonad morphogenesis in an activity-dependent and DTC cell-autonomous manners. In the src-1 mutants, DTCs fail to turn and continue their centrifugal migration along the ventral muscles. The effect of the src-1 mutation is suppressed by mutations in genes that function in the CED/Rac pathway, suggesting that SRC-1 in DTCs is an upstream regulator of a Rac pathway that controls cytoskeletal remodeling. In the src-1 mutant, the expression of unc-5/netrin receptor is normally regulated, and neither the precocious expression of UNC-5 nor the mutation in the unc-5 gene significantly affects the DTC migration defect. These data suggest that SRC-1 acts in the netrin signaling in DTCs. The src-1 mutant also exhibits cell-autonomous defects in the migration and growth cone path-finding of Q neuroblast descendants AVM and PVM. However, these roles of SRC-1 do not appear to involve the CED/Rac pathway. These findings show that SRC-1 functions in responding to various extracellular guidance cues that direct the cell migration via disparate signaling pathways in different cell types.     

Lipid Droplet Protein LID-1 Mediates ATGL-1-Dependent Lipolysis during Fasting in Caenorhabditis elegans

Lipolysis is a delicate process involving complex signaling cascades and sequential enzymatic activations. In Caenorhabditis elegans, fasting induces various physiological changes, including a dramatic decrease in lipid contents through lipolysis. Interestingly, C. elegans lacks perilipin family genes which play a crucial role in the regulation of lipid homeostasis in other species. Here, we demonstrate that in the intestinal cells of C. elegans, a newly identified protein, lipid droplet protein 1 (C25A1.12; LID-1), modulates lipolysis by binding to adipose triglyceride lipase 1 (C05D11.7; ATGL-1) during nutritional deprivation. In fasted worms, lipid droplets were decreased in intestinal cells, whereas suppression of ATGL-1 via RNA interference (RNAi) resulted in retention of stored lipid droplets. Overexpression of ATGL-1 markedly decreased lipid droplets, whereas depletion of LID-1 via RNAi prevented the effect of overexpressed ATGL-1 on lipolysis. In adult worms, short-term fasting increased cyclic AMP (cAMP) levels, which activated protein kinase A (PKA) to stimulate lipolysis via ATGL-1 and LID-1. Moreover, ATGL-1 protein stability and LID-1 binding were augmented by PKA activation, eventually leading to increased lipolysis. These data suggest the importance of the concerted action of lipase and lipid droplet protein in the response to fasting signals via PKA to maintain lipid homeostasis.     

FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association of SR to intracellular membranes. Our data highlight an atypical role of FBXO22 in enhancing d-serine synthesis that is unrelated to its classical effects as a component of the ubiquitin-proteasome degradation pathway.     

RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.     

BRIZ1 and BRIZ2 Proteins Form a Heteromeric E3 Ligase Complex Required for Seed Germination and Post-germination Growth in Arabidopsis thaliana

Ubiquitin pathway E3 ligases are an important component conferring specificity and regulation in ubiquitin attachment to substrate proteins. The Arabidopsis thaliana RING (Really Interesting New Gene) domain-containing proteins BRIZ1 and BRIZ2 are essential for normal seed germination and post-germination growth. Loss of either BRIZ1 (At2g42160) or BRIZ2 (At2g26000) results in a severe phenotype. Heterozygous parents produce progeny that segregate 3:1 for wild-type:growth-arrested seedlings. Homozygous T-DNA insertion lines are recovered for BRIZ1 and BRIZ2 after introduction of a transgene containing the respective coding sequence, demonstrating that disruption of BRIZ1 or BRIZ2 in the T-DNA insertion lines is responsible for the observed phenotype. Both proteins have multiple predicted domains in addition to the RING domain as follows: a BRAP2 (BRCA1-Associated Protein 2), a ZnF UBP (Zinc Finger Ubiquitin Binding protein), and a coiled-coil domain. In vitro, both BRIZ1 and BRIZ2 are active as E3 ligases but only BRIZ2 binds ubiquitin. In vitro synthesized and purified recombinant BRIZ1 and BRIZ2 preferentially form hetero-oligomers rather than homo-oligomers, and the coiled-coil domain is necessary and sufficient for this interaction. BRIZ1 and BRIZ2 co-purify after expression in tobacco leaves, which also requires the coiled-coil domain. BRIZ1 and BRIZ2 coding regions with substitutions in the RING domain are inactive in vitro and, after introduction, fail to complement their respective mutant lines. In our current model, BRIZ1 and BRIZ2 together are required for formation of a functional ubiquitin E3 ligase in vivo, and this complex is required for germination and early seedling growth.     

Bioengineered Chinese Hamster Ovary Cells with Golgi-targeted 3-O-Sulfotransferase-1 Biosynthesize Heparan Sulfate with an Antithrombin-binding Site

HS3st1 (heparan sulfate 3-O-sulfotransferase isoform-1) is a critical enzyme involved in the biosynthesis of the antithrombin III (AT)-binding site in the biopharmaceutical drug heparin. Heparin is a highly sulfated glycosaminoglycan that shares a common biosynthetic pathway with heparan sulfate (HS). Although only granulated cells, such as mast cells, biosynthesize heparin, all animal cells are capable of biosynthesizing HS. As part of an effort to bioengineer CHO cells to produce heparin, we previously showed that the introduction of both HS3st1 and NDST2 (N-deacetylase/N-sulfotransferase isoform-2) afforded HS with a very low level of anticoagulant activity. This study demonstrated that untargeted HS3st1 is broadly distributed throughout CHO cells and forms no detectable AT-binding sites, whereas Golgi-targeted HS3st1 localizes in the Golgi and results in the formation of a single type of AT-binding site and high anti-factor Xa activity (137 ± 36 units/mg). Moreover, stable overexpression of HS3st1 also results in up-regulation of 2-O-, 6-O-, and N-sulfo group-containing disaccharides, further emphasizing a previously unknown concerted interplay between the HS biosynthetic enzymes and suggesting the need to control the expression level of all of the biosynthetic enzymes to produce heparin in CHO cells.     

Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.