Serine-rich Motif Research Articles

Capturing Autoinhibited PDK1 Reveals the Linker's Regulatory Role, Informing Innovative Inhibitor Design.

PDK1 is crucial for PI3K/AKT/mTOR and Ras/MAPK cancer signaling. It phosphorylates AKT in a PIP3-dependent but S6K, SGK, and RSK kinases in a PIP3-independent manner. Unlike its substrates, its autoinhibited monomeric state has been unclear, likely due to its low population time, and phosphorylation in the absence of PIP3 has been puzzling too. Here, guided by experimental data, we constructed models and performed all-atom molecular dynamics simulations. In the autoinhibited PDK1 conformation that resembles autoinhibited AKT, binding of the linker between the kinase and PH domains to the PIF-binding pocket promotes the formation of the Glu130-Lys111 salt bridge and weakens the association of the kinase domain with the PH domain, shifting the population from the autoinhibited state to states accessible to the membrane and its kinase substrates. The interaction of the substrates' hydrophobic motif and the PDK1 PIF-binding pocket facilitates the release of the autoinhibition even in the absence of PIP3. Phosphorylation of the serine-rich motif within the linker further attenuates the association of the PH domain with the kinase domain. These suggest that while the monomeric autoinhibited state is relatively stable, it can readily shift to its active, catalysis-prone state to phosphorylate its diverse substrates. Our findings reveal the PDK1 activation mechanism and discover the regulatory role of PDK1's linker, which lead to two innovative linker-based inhibitor strategies: (i) locking the autoinhibited PDK1 through optimization of the interactions of AKT inhibitors with the PH domain of PDK1 and (ii) analogs (small molecules or peptidomimetics) that mimic the linker interactions with the PIF-binding pocket.

Proteomic characterization of extracellular vesicles derived from lactic acid bacteria.

Lactobacillus species confer health benefits by their metabolites, secreted molecules, and population numbers. Extracellular vesicles (EVs) are nano-sized particles released from cells and mediate intercellular communications. EVs-encapsulated cargos are a crucial key to decide involved biological function. However, little is known about the composition of EVs, leaving mechanisms by which Lactobacillus-derived EVs affect recipient cells remaining unresolved. This study examined the composition of EV proteins from Lactobacillus species by using liquid chromatography coupled with tandem mass spectrometry, including L. plantarum, L. fermentum, and L. gasseri. The major proteins of EVs are associated with biological processes such as catalytic activity, gluco-neogenesis, cell wall organization, and glycolytic processes. Motif enrichment analysis revealed that EVs from L. plantarum and L. fermentum contained proteins with serine-rich motif. This is the first study to report the composition and comparison of EV proteins from Lactobacillus species, providing important information of EVs in functional food products development.

Variation and Selection in the Putative Sperm-Binding Region of ZP3 in Muroid Rodents: A Comparison between Cricetids and Murines

In mammals, the zona pellucida glycoprotein 3 (ZP3) is considered a primary sperm receptor of the oocyte and is hypothesized to be involved in reproductive isolation. We investigated patterns of diversity and selection in the putative sperm-binding region (pSBR) of mouse ZP3 across Cricetidae and Murinae, two hyperdiverse taxonomic groups within muroid rodents. In murines, the pSBR is fairly conserved, in particular the serine-rich stretch containing the glycosylation sites proposed as essential for sperm binding. In contrast, cricetid amino acid sequences of the pSBR were much more variable and the serine-rich motif, typical of murines, was generally substantially modified. Overall, our results suggest a general lack of species specificity of the pSBR across the two muroid families. We document statistical evidence of positive selection acting on exons 6 and 7 of ZP3 and identified several amino acid sites that are likely targets of selection, with most positively selected sites falling within or adjacent to the pSBR.

Cloning and cold-resistance analyses of CfICE1 gene in Cryptomeria fortunei

Cryptomeria fortunei is a conifer species that can attain a height of ~70 m and is cultivated for its timber as well as its ornamental value. It is a subtropical plant that prefers a warm and humid environment. Therefore, low temperature (LT) affects its growth, development, productivity and ecological distribution. Inducer of C-repeat binding factor (CBF) expression 1 (ICE1) plays an important role in the response to cold/freezing stress in plants through the CBF regulation pathway. To date, there is no research on homologue of ICE1 in C. fortunei. In this study, we first isolated the CfICE1 transcript from C. fortunei. The CfICE1 coding sequence was 1728 nucleotides encoding a 575-aa protein and contained a serine-rich motif, a basic helix-loop-helix-Zipper (bHLH-Zip), an ACT domain and a nuclear localization signal (NLS), which were conserved in ICE1 homologous genes. Phylogenetic analysis showed that CfICE1 and all dicots ICE1 proteins were clustered together. CfICE1 had transcriptional activity in yeast cells, was predominantly located in the nucleus and highly expressed in tender needles and roots. 35S::CfICE1 transgenic Arabidopsis thaliana could increase antioxidant enzyme activities and photosynthesis and reduce the malondialdehyde content compared to the wild-type to better cope with LT. Under LT, CfICE1 expression was higher; the C. fortunei clone with stronger cold resistance (CR) significantly upregulated the expression of CfICE1 compared to the weaker clone. In conclusion, these results suggest that CfICE1 plays an active role in CR, which provides a theoretical basis for breeding for CR in C. fortunei.

Phospho-regulation of the Shugoshin - Condensin interaction at the centromere in budding yeast.

Correct bioriented attachment of sister chromatids to the mitotic spindle is essential for chromosome segregation. In budding yeast, the conserved protein shugoshin (Sgo1) contributes to biorientation by recruiting the protein phosphatase PP2A-Rts1 and the condensin complex to centromeres. Using peptide prints, we identified a Serine-Rich Motif (SRM) of Sgo1 that mediates the interaction with condensin and is essential for centromeric condensin recruitment and the establishment of biorientation. We show that the interaction is regulated via phosphorylation within the SRM and we determined the phospho-sites using mass spectrometry. Analysis of the phosphomimic and phosphoresistant mutants revealed that SRM phosphorylation disrupts the shugoshin–condensin interaction. We present evidence that Mps1, a central kinase in the spindle assembly checkpoint, directly phosphorylates Sgo1 within the SRM to regulate the interaction with condensin and thereby condensin localization to centromeres. Our findings identify novel mechanisms that control shugoshin activity at the centromere in budding yeast.

Casein Kinase 1 Regulates Cytorhabdovirus Replication and Transcription by Phosphorylating a Phosphoprotein Serine-Rich Motif.

Casein kinase 1 (CK1) family members are conserved Ser/Thr protein kinases that regulate important developmental processes in all eukaryotic organisms. However, the functions of CK1 in plant immunity remain largely unknown. Barley yellow striate mosaic virus (BYSMV), a plant cytorhabdovirus, infects cereal crops and is obligately transmitted by the small brown planthopper (SBPH; Laodelphax striatellus). The BYSMV phosphoprotein (P) exists as two forms with different mobilities corresponding to 42 kD (P42) and 44 kD (P44) in SDS-PAGE gels. Mass spectrometric analyses revealed a highly phosphorylated serine-rich (SR) motif at the C-terminal intrinsically disordered region of the P protein. The Ala-substitution mutant (PS5A) in the SR motif stimulated virus replication, whereas the phosphorylation-mimic mutant (PS5D) facilitated virus transcription. Furthermore, PS5A and PS5D associated preferentially with nucleocapsid protein-RNA templates and the large polymerase protein to provide optimal replication and transcription complexes, respectively. Biochemistry assays demonstrated that plant and insect CK1 protein kinases could phosphorylate the SR motif and induce conformational changes from P42 to P44. Moreover, overexpression of CK1 or a dominant-negative mutant impaired the balance between P42 and P44, thereby compromising virus infections. Our results demonstrate that BYSMV recruits the conserved CK1 kinases to achieve its cross-kingdom infection in host plants and insect vectors.

Recombinant expression of sericin-cecropin fusion protein and its functional activity.

Silk sericin is a natural polymer with potential utility in biomedical and biotechnological applications. Recombinantly expressed sericin ensures a source of pure protein with no contamination and with multiple properties when expressed as a fusion protein. Hence, the present paper aims to recombinantly express a functional silk sericin fusion protein. In order to develop a more effective sericin protein, we have attempted to recombinantly express a part of sericin sequence, which represents a highly conserved and internally repetitive unit of the sericin1 protein, and its fusion with cecropin B, a potent antimicrobial peptide. Both difficult-to-express proteins were expressed in Escherichia coli and purified by nickel-charged affinity resin. Further, functional assay demonstrated that both proteins were individually active against Gram-positive and negative bacteria, with enhanced bactericidal activity observed in sericin-cecropin B fusion protein. To our knowledge, this is the first report not only on the recombinant expression of sericin as a fusion protein but also the bactericidal possibility of the 38-amino acid serine-rich motif of sericin protein. We also discuss the potential biomedical and biotechnological applications of this sericin hybrid protein.

An essential N-terminal serine-rich motif in the AAV VP1 and VP2 subunits that may play a role in viral transcription

Adeno-associated virus (AAV) is one of the most researched, clinically utilized gene therapy vectors. Though clinical success has been achieved, transgene delivery and expression may be hindered by cellular and tissue barriers. Understanding the role of receptor binding, entry, endosomal escape, cytoplasmic and nuclear trafficking, capsid uncoating, and viral transcription in therapeutic efficacy is paramount. Previous studies have shown that N-terminal regions of the AAV capsid proteins are responsible for endosomal escape and nuclear trafficking, however the mechanisms remain unknown. We identified a highly-conserved three-residue serine/threonine (S/T) motif in the capsid N-terminus, previously uncharacterized in its role in intracellular trafficking and transduction. Using alanine scanning mutagenesis, we found S155 and the flanking residues, D154 and G158, are essential for AAV2 transduction efficiency. Remarkably, specific capsid mutants show a 5 to 9-fold decrease in viral mRNA transcripts, highlighting a potential role of the S/T motif in transcription of the viral genome.

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this study we show that isolated PH and START domains interact with each other. The crystal structure of a PH-START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH-START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine-rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.

GSK-3-mediated phosphorylation couples ER-Golgi transport and nuclear stabilization of the CREB-H transcription factor to mediate apolipoprotein secretion.

CREB-H, an ER-anchored transcription factor, plays a key role in regulating secretion in metabolic pathways, particularly triglyceride homeostasis. It controls the production both of secretory pathway components and cargoes, including apolipoproteins ApoA-IV and ApoC-II, contributing to VLDL/HDL distribution and lipolysis. The key mechanism controlling CREB-H activity involves its ER retention and forward transport to the Golgi, where it is cleaved by Golgi-resident proteases, releasing the N-terminal product, which traffics to the nucleus to effect transcriptional responses. Here we show that a serine-rich motif termed the P-motif, located in the N-terminus between serines 73 and 90, controls release of the precursor transmembrane form from the ER and its forward transport to the Golgi. This motif is subject to GSK-3 phosphorylation, promoting ER retention, while mutation of target serines and drug inhibition of GSK-3 activity coordinately induce both forward transport of the precursor and cleavage, resulting in nuclear import. We previously showed that for the nuclear product, the P-motif is subject to multiple phosphorylations, which regulate stability by targeting the protein to the SCFFbw1a E3 ubiquitin ligase. Thus phosphorylation at the P-motif provides integrated control of CREB-H function, coupling intercompartmental transport in the cytoplasm with stabilization of the active form in the nucleus.

Correlated evolution between CK1δ Protein and the Serine-rich Motif Contributes to Regulating the Mammalian Circadian Clock

Understanding the mechanism underlying the physiological divergence of species is a long-standing issue in evolutionary biology. The circadian clock is a highly conserved system existing in almost all organisms that regulates a wide range of physiological and behavioral events to adapt to the day-night cycle. Here, the interactions between hCK1ϵ/δ/DBT (Drosophila ortholog of CK1δ/ϵ) and serine-rich (SR) motifs from hPER2 (ortholog of Drosophila per) were reconstructed in a Drosophila circadian system. The results indicated that in Drosophila, the SR mutant form hPER2S662G does not recapitulate the mouse or human mutant phenotype. However, introducing hCK1δ (but not DBT) shortened the circadian period and restored the SR motif function. We found that hCK1δ is catalytically more efficient than DBT in phosphorylating the SR motif, which demonstrates that the evolution of CK1δ activity is required for SR motif modulation. Moreover, an abundance of phosphorylatable SR motifs and the striking emergence of putative SR motifs in vertebrate proteins were observed, which provides further evidence that the correlated evolution between kinase activity and its substrates set the stage for functional diversity in vertebrates. It is possible that such correlated evolution may serve as a biomarker associated with the adaptive benefits of diverse organisms. These results also provide a concrete example of how functional synthesis can be achieved through introducing evolutionary partners in vivo.

A small part of myosin IIB takes on a big role in cell polarity.

A migrating cell must establish front-to-back polarity in order to move. In this issue, Juanes-Garcia et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201407059) report that a short serine-rich motif in nonmuscle myosin IIB is required to establish the cell’s rear. This motif represents a new paradigm for what determines directional cell migration.

Phosphoregulation of the Ceramide Transport Protein CERT at Serine 315 in the Interaction with VAMP-associated Protein (VAP) for Inter-organelle Trafficking of Ceramide in Mammalian Cells

The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We show that the phosphorylation of CERT at serine 315, which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at Ser-315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine-rich motif) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the serine-rich motif and the phosphorylation of Ser-315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.

Reversible Assembly of β-Sheet Nanocrystals within Caddisfly Silk

Nuclear magnetic resonance (NMR) and X-ray diffraction (XRD) experiments reveal the structural importance of divalent cation-phosphate complexes in the formation of β-sheet nanocrystals from phosphorylated serine-rich regions within aquatic silk from caddisfly larvae of the species Hesperophyla consimilis. Wide angle XRD data on native caddisfly silk show that the silk contains a significant crystalline component with a repetitive orthorhombic unit cell aligned along the fiber axis with dimensions of 5.9 Å × 23.2 Å × 17.3 Å. These nanocrystalline domains depend on multivalent cations, which can be removed through chelation with ethylenediaminetetraacetic acid (EDTA). A comparison of wide angle X-ray diffraction data before and after EDTA treatment reveals that the integrated peak area of reflections corresponding to the nanocrystalline regions decreases by 15-25% while that of the amorphous background reflections increases by 20%, indicating a partial loss of crystallinity. (31)P solid-state NMR data on native caddisfly silk also show that the phosphorylated serine-rich motifs transform from a rigid environment to one that is highly mobile and water-solvated after treatment with EDTA. The removal of divalent cations through exchange and chelation has therefore caused a collapse of the β-sheet structure. However, NMR results show that the rigid phosphorus environment is mostly recovered after the silk is re-treated with calcium. The (31)P spin-lattice (T1) relaxation times were measured at 7.6 ± 3.1 and 1 ± 0.5 s for this calcium-recovered sample and the native silk sample, respectively. The shorter (31)P T1 relaxation times measured for the native silk sample are attributed to the presence of paramagnetic iron that is stripped away during EDTA chelation treatment and replaced with diamagnetic calcium.

Structural Characterization of Caddisfly Silk with Solid-State NMR and X-Ray Diffraction

Adhesive silks spun by aquatic caddisfly (order Trichoptera) larvae are used to build both intricate protective shelters and food harvesting nets underwater. In this study, we use 13C and 31P solid-state NMR and wide angle X-ray diffraction (WAXD) as tools to elucidate molecular protein structure of caddisfly larval silk. Caddisfly silk is a fibroin protein-based biopolymer containing mostly repetitive amino acid motifs. Solid-state NMR results provide strong evidence for a structural model where phosphorylated serine repeats (pSX)4 complex with di- and trivalent cations Ca2+, Mg2+ and Fe3+ to form rigid nanocrystalline β-sheet structures in caddisfly silk (Addison et al., Biomacromolecules (2013)). NMR data suggests that both phosphorylated serine and neighboring valine residues exist in a β-sheet conformation while glycine and leucine residues common in GGX repeats reside in random coil conformations. 31P chemical shift anisotropy (CSA) powder pattern analysis of native caddisfly silk indicates that the phosphates on phosphoserine residues are rigid, doubly ionized, and charge-stabilized by divalent cations. 31P solid-state NMR data also shows that the phosphorylated serine-rich motifs transform from a rigid environment to one that is highly mobile and water-solvated after treatment with the metal ion chelator, EDTA. However, the rigid phosphorus environment is mostly recovered after the silk is retreated with calcium. While a strong interaction between negatively charged phosphates and multivalent cations is no surprise, its prevalence within caddisfly larval silk is unique within silk-based biopolymers. In this work we demonstrate that multivalent cations are absolutely essential to the structural integrity of caddisfly silk.

Functional Conservation and Structural Diversification of Silk Sericins in Two Moth Species

Sericins are hydrophilic structural proteins produced by caterpillars in the middle section of silk glands and layered over fibroin proteins secreted in the posterior section. In the process of spinning, fibroins form strong solid filaments, while sericins seal the pair of filaments into a single fiber and glue the fiber into a cocoon. Galleria mellonella and the previously examined Bombyx mori harbor three sericin genes that encode proteins containing long repetitive regions. Galleria sericin genes are similar to each other and the protein repeats are built from short and extremely serine-rich motifs, while Bombyx sericin genes are diversified and encode proteins with long and complex repeats. Developmental changes in sericin properties are controlled at the level of gene expression and splicing. In Galleria , MG-1 sericin is produced throughout larval life until the wandering stage, while the production of MG-2 and MG-3 reaches a peak during cocoon spinning.

Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis

The endoplasmic reticulum (ER)-Golgi sterol transfer activity of oxysterol-binding protein (OSBP) regulates sphingomyelin (SM) synthesis, as well as post-Golgi cholesterol efflux pathways. The phosphorylation and ER-Golgi localization of OSBP are correlated, suggesting this modification regulates the directionality and/or specificity of transfer activity. In this paper, we report that phosphorylation on two serine-rich motifs, S381-S391 (site 1) and S192, S195, S200 (site 2), specifically controls OSBP activity at the ER. A phosphomimetic of the SM/cholesterol-sensitive phosphorylation site 1 (OSBP-S5E) had increased in vitro cholesterol and 25-hydroxycholesterol-binding capacity, and cholesterol extraction from liposomes, but reduced transfer activity. Phosphatidylinositol 4-phosphate (PI(4)P) and cholesterol competed for a common binding site on OSBP; however, direct binding of PI(4)P was not affected by site 1 phosphorylation. Individual site 1 and site 2 phosphomutants supported oxysterol activation of SM synthesis in OSBP-deficient CHO cells. However, a double site1/2 mutant (OSBP-S381A/S3D) was deficient in this activity and was constitutively colocalized with vesicle-associated membrane protein-associated protein A (VAP-A) in a collapsed ER network. This study identifies phosphorylation regulation of sterol and VAP-A binding by OSBP in the ER, and PI(4)P as an alternate ligand that could be exchanged for sterol in the Golgi apparatus.

CD69 Suppresses Sphingosine 1-Phosophate Receptor-1 (S1P1) Function through Interaction with Membrane Helix 4

Lymphocyte egress from lymph nodes requires the G-protein-coupled sphingosine 1-phosphate receptor-1 (S1P1). The activation antigen CD69 associates with and inhibits the function of S1P1, inhibiting egress. Here we undertook biochemical characterization of the requirements for S1P1-CD69 complex formation. Domain swapping experiments between CD69 and the related type II transmembrane protein, NKRp1A, identified a requirement for the transmembrane and membrane proximal domains for specific interaction. Mutagenesis of S1P1 showed a lack of requirement for N-linked glycosylation, tyrosine sulfation, or desensitization motifs but identified a requirement for transmembrane helix 4. Expression of CD69 led to a reduction of S1P1 in cell lysates, likely reflecting degradation. Unexpectedly, the S1P1-CD69 complex exhibited a much longer half-life for binding of S1P than S1P1 alone. In contrast to wild-type CD69, a non-S1P1 binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S1P1 and suggest that CD69 induces an S1P1 conformation that shares some properties of the ligand-bound state, thereby facilitating S1P1 internalization and degradation.

N-terminal PH domain and C-terminal auto-inhibitory region of CKIP-1 coordinate to determine its nucleus-plasma membrane shuttling

The pleckstrin homology (PH) domain-containing protein casein kinase 2 interacting protein-1 (CKIP-1) plays an important role in regulation of bone formation and muscle differentiation. How CKIP-1 localization is determined remains largely unclear. We observed that isolated CKIP-1-PH domain was predominantly localized in the nucleus and the C-terminus of CKIP-1 counteracted its nuclear localization. The net charge of basic residues and a serine-rich motif within the PH domain plays a pivotal role in the localization switch of both full-length CKIP-1 and the isolated PH domain. We propose that the N-terminal PH domain and C-terminal auto-inhibitory region of CKIP-1 coordinate to determine its subcellular localization and the nucleus–plasma membrane shuttling.

Sprouty proteins: modified modulators, matchmakers or missing links?

Sprouty proteins are involved in organogenesis, particularly during the branching of endothelial tubes, and existing evidence suggests that Sprouty's point of action lies downstream of receptor signaling to inhibit the activation of the central Ras/Erk pathway. How Sprouty proteins accomplish their inhibitory action and whether they interact with other signaling pathways are significant questions. Sprouty proteins are devoid of any recognizable protein interaction domain, and clues as to how they function have been mainly derived from screening for interacting partners. Conserved across all the Sprouty proteins are three sequences: a Cbl-tyrosine kinase-binding (TKB) binding motif centered on an obligatorily phosphorylated tyrosine (Y55 in Sprouty2), a serine-rich motif (SRM) and a cysteine-rich domain (CRD). With the exception of a handful of proteins that bind to the N-terminus, most of the binding to Sprouty occurs via the CRD, predominantly by serine/threonine kinases that target sites within the SRM on Sprouty. Some of the resultant increase in phosphorylation is opposed by activated protein phosphatase 2A that binds to the N-terminal Cbl-TKB binding motif. Significantly, two ubiquitin E3 ligases also bind to the N-terminus of Sprouty: c-Cbl binds with high affinity to the TKB binding motif and SIAH2 binds constitutively to a different site; both proteins are able to direct the ubiquitination of Sprouty proteins and its destruction. The collective evidence points to Sprouty proteins as being substantially covalently-modified to control its location, stability, association, and destruction. With such stringent control of the Sproutys, the main question is what key proteins does this facilitator bring together?