Characterization of early and late transition states of the folding pathway of a SH2 domain

Albeit SH2 domains are abundant protein–protein interaction modules with fundamental roles in the regulation of several physiological and molecular pathways in the cell, the available information about the determinants of their thermodynamic stability and folding properties are still very limited. In this work, we provide a quantitative characterization of the folding pathway of the C‐terminal SH2 domain of SHP2, conducted through a combination of site‐directed mutagenesis and kinetic (un)folding experiments (Φ‐value analysis). The energetic profile of the folding reaction of the C‐SH2 domain is described by a three‐state mechanism characterized by the presence of two transition states and a high‐energy intermediate. The production of 29 site‐directed variants allowed us to calculate the degree of native‐like interactions occurring in the early and late events of the folding reaction. Data analysis highlights the presence of a hydrophobic folding nucleus surrounded by a lower degree of structure in the early events of folding, further consolidated as the reaction proceeds towards the native state. Interestingly, residues physically located in the functional region of the domain reported unusual Φ‐values, a hallmark of the presence of transient misfolding. We compared our results with previous ones obtained for the N‐terminal SH2 domain of SHP2. Notably, a conserved complex folding mechanism implying the presence of a folding intermediate arise from comparison, and the relative stability of such intermediate appears to be highly sequence dependent. Data are discussed under the light of previous works on SH2 domains.     

SARS‐CoV‐2 nucleocapsid protein phase‐separates with RNA and with human hnRNPs

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA‐binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) condenses with RNA via liquid–liquid phase separation (LLPS) and that N protein can be recruited in phase‐separated forms of human RNA‐binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N‐terminal IDR and central‐linker IDR, as well as the folded C‐terminal oligomerization domain, while the folded N‐terminal domain and the C‐terminal IDR are not required. N protein phase separation is induced by addition of non‐specific RNA. In addition, N partitions in vitro into phase‐separated forms of full‐length human hnRNPs (TDP‐43, FUS, hnRNPA2) and their low‐complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS‐CoV‐2 viral genome packing and in host‐protein co‐opting necessary for viral replication and infectivity.     

Phosphotyrosines 627 and 659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) conferring binding and activation of SHP2

A major Grb2-associated binder-1 (Gab1) binding partner in epidermal growth factor (EGF)-stimulated cells is protein-tyrosine phosphatase (PTPase) SHP2, which contains tandem SH2 domains. The SHP2 PTPase activity is required for activation of the extracellular signal-regulated kinase (ERK) subfamily of mitogen-activated protein (MAP) kinase by EGF. To investigate the mechanism by which Gab1 and SHP2 mediate ERK activation, we characterized the Gab1-SHP2 interaction. We found that both Tyr-627 and Tyr-659 of Gab1 were required for SHP2 binding to Gab1 and for ERK2 activation by EGF. Far Western blot analysis suggested that the tandem SH2 domains of SHP2 bind to Gab1 in a specific orientation, in which the N-SH2 domain binds to phosphotyrosine (Tyr(P))-627 and the C-SH2 domain binds to Tyr(P)-659. When assayed with peptide substrates, SHP2 PTPase was activated by a bisphosphopeptide containing both Tyr(P)-627 and Tyr(P)-659, but not by monophosphopeptides containing Tyr(P)-627 or Tyr(P)-659 or a mixture of these monophosphopeptides. These results suggest that Tyr(P)-627 and Tyr(P)-659 of Gab1 constitute a bisphosphoryl tyrosine-based activation motif (BTAM) that binds and activates SHP2. Remarkably, while a constitutively active SHP2 (SHP2DeltaN) could not rescue the defect of a SHP2-binding defective Gab1 (Gab1FF) in ERK2 activation, expression of a Gab1FF-SHP2DeltaN chimera resulted in constitutive activation of ERK2 in transfected cells. Thus, physical association of activated SHP2 with Gab1 is necessary and sufficient to mediate the ERK mitogen-activated protein kinase activation. Phosphopeptides derived from Gab1 were dephosphorylated by active SHP2 in vitro. Consistently, substrate-trapping experiments with a SHP2 catalytic inactive mutant suggested that Gab1 was a SHP2 PTPase substrate in the cells. Therefore, Gab1 not only is a SHP2 activator but also is a target of its PTPase.     

Completing the family of human Eps15 homology domains: Solution structure of the internal Eps15 homology domain of γ‐synergin

Eps15 homology (EH) domains are universal interaction domains to establish networks of protein–protein interactions in the cell. These networks mainly coordinate cellular functions including endocytosis, actin remodeling, and other intracellular signaling pathways. They are well characterized in structural terms, except for the internal EH domain from human γ‐synergin (EHγ). Here, we complete the family of EH domain structures by determining the solution structure of the EHγ domain. The structural ensemble follows the canonical EH domain fold and the identified binding site is similar to other known EH domains. But EHγ differs significantly in the N‐ and C‐terminal regions. The N‐terminal α‐helix is shortened compared to known homologues, while the C‐terminal one is fully formed. A significant proportion of the remaining N‐ and C‐terminal regions are well structured, a feature not seen in other EH domains. Single mutations in both the N‐terminal and the C‐terminal structured extensions lead to the loss of the distinct three‐dimensional fold and turn EHγ into a molten globule like state. Therefore, we propose that the structural extensions in EHγ function as a clamp and are undoubtedly required to maintain its tertiary fold.     

Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor

Grb2-associated binder-1 (Gab1) is a multisite docking protein containing a pleckstrin homology (PH) domain, multiple potential tyrosine phosphorylation sites, and several proline-rich sequences. Gab1 becomes tyrosine-phosphorylated in cells stimulated with growth factors, cytokines, and ligands for G protein-coupled receptors. A major Gab1-binding protein detected in cells treated with extracellular stimuli is the tyrosine phosphatase, SHP2. Although the role of SHP2-Gab1 interaction in cell signaling has not yet been characterized, SHP2 is known to mediate mitogen-activated protein (MAP) kinase activation induced by the epidermal growth factor (EGF). However, the mechanism by which the SHP2 phosphatase exerts a positive signaling role remains obscure. In this study, we prepared Gab1 mutants lacking the SHP2 binding site (Gab1Y627F), the phosphatidylinositol 3-kinase (PI3K) binding sites (Gab1DeltaPI3K), and the PH domain (Gab1DeltaPH). Expression of Gab1Y627F blocked the extracellular signal-regulated kinase-2 (ERK2) activation by lysophosphatidic acid (LPA) and EGF. Conversely, expression of the wild-type Gab1 in HEK293 cells augmented the LPA receptor Edg2-mediated ERK2 activation. Whereas the PH domain was required for Gab1 mediation of ERK2 activation by LPA, it was not essential for EGF-induced ERK2 activation. Expression of Gab1DeltaPI3K had no apparent effect on ERK2 activation by LPA and EGF in the cells that we have examined. These results establish a role for Gab1 in the LPA-induced MAP kinase pathway and clearly demonstrate that Gab1-SHP2 interaction is essential for ERK2 activation by LPA and EGF. These findings also suggest that the positive role of SHP2 in the MAP kinase pathway depends on its interaction with Gab1.     

Engagement of Gab1 and Gab2 in erythropoietin signaling.

Several signaling cascades are activated during engagement of the erythropoietin receptor to mediate the biological effects of erythropoietin. The members of the insulin receptor substrate (IRS) family of proteins play a central role in signaling for various growth factor receptors and cytokines by acting as docking proteins for the SH2 domains of signaling elements, linking cytokine receptors to diverse downstream pathways. In the present study we provide evidence that the recently cloned IRS-related proteins, Gab1 and Gab2, of the Gab family of proteins, are rapidly phosphorylated on tyrosine during erythropoietin treatment of erythropoietin-responsive cells and provide docking sites for the engagement of the SHP2 phosphatase and the p85 subunit of the phosphatidylinositol 3'-kinase. Furthermore, our data show that Gab1 is the primary IRS-related protein activated by erythropoietin in primary erythroid progenitor cells. In studies to identify the erythropoietin receptor domains required for activation of Gab proteins, we found that tyrosines 425 and 367 in the cytoplasmic domain of the erythropoietin receptor are required for the phosphorylation of Gab2. Taken together, our data demonstrate that Gab proteins are engaged in erythropoietin signaling to mediate downstream activation of the SHP2 and phosphatidylinositol 3'-kinase pathways and possibly participate in the generation of the erythropoietin-induced mitogenic responses.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Cyclic nucleotide selectivity of protein kinase G isozymes

The intrinsic activity of the C‐terminal catalytic (C) domain of cyclic guanosine monophosphate (cGMP)‐dependent protein kinases (PKG) is inhibited by interactions with the N‐terminal regulatory (R) domain. Selective binding of cGMP to cyclic nucleotide binding (CNB) domains within the R‐domain disrupts the inhibitory R–C interaction, leading to the release and activation of the C‐domain. Affinity measurements of mammalian and plasmodium PKG CNB domains reveal different degrees of cyclic nucleotide affinity and selectivity; the CNB domains adjacent to the C‐domain are more cGMP selective and therefore critical for cGMP‐dependent activation. Crystal structures of isolated CNB domains in the presence and absence of cyclic nucleotides reveal isozyme‐specific contacts that explain cyclic nucleotide selectivity and conformational changes that accompany CNB. Crystal structures of tandem CNB domains identify two types of CNB‐mediated dimeric contacts that indicate cGMP‐driven reorganization of domain–domain interfaces that include large conformational changes. Here, we review the available structural and functional information of PKG CNB domains that further advance our understanding of cGMP mediated regulation and activation of PKG isozymes.     

The novel role of the C-terminal region of SHP-2. Involvement of Gab1 and SHP-2 phosphatase activity in Elk-1 activation

SHP-2, a nontransmembrane-type protein-tyrosine phosphatase that contains two Src homology 2 (SH2) domains, is thought to participate in growth factor signal transduction pathways via SH2 domain interactions. To determine the role of each region of SHP-2 in platelet-derived growth factor signaling assayed by Elk-1 activation, we generated six deletion mutants of SHP-2. The large SH2 domain deletion SHP-2 mutant composed of amino acids 198-593 (SHP-2-(198-593)), but not the smaller SHP-2-(399-593), showed significantly higher SHP-2 phosphatase activity in vitro. In contrast, SHP-2-(198-593) mutant inhibited wild type SHP-2 phosphatase activity, whereas SHP-2-(399-593) mutant increased activity. To understand these functional changes, we focused on the docking protein Gab1 that assembles signaling complexes. Pull-down experiments with Gab1 suggested that the C-terminal region of SHP-2 as well as the SH2 domains (N-terminal region) associated with Gab1, but the SHP-2-(198-593) mutant did not associate with Gab1. SHP-2-(1-202) or SHP-2-(198-593) inhibited platelet-derived growth factorinduced Elk-1 activation, but SHP-2-(399-593) increased Elk-1 activation. Co-expression of SHP-2-(1-202) with SHP-2-(399-593) inhibited SHP-2-(399-593)/Gab1 interaction, and the SHP-2-(399-593) mutant induced SHP-2 phosphatase and Elk-1 activation, supporting the autoinhibitory effect of SH2 domains on the C-terminal region of SHP-2. These data suggest that both SHP-2/Gab1 interaction in the C-terminal region of SHP-2 and increased SHP-2 phosphatase activity are important for Elk-1 activation. Furthermore, we identified a novel sequence for SHP-2/Gab1 interactions in the C-terminal region of SHP-2.     

The C‐degron pathway eliminates mislocalized proteins and products of deubiquitinating enzymes

Protein termini are determinants of protein stability. Proteins bearing degradation signals, or degrons, at their amino‐ or carboxyl‐termini are eliminated by the N‐ or C‐degron pathways, respectively. We aimed to elucidate the function of C‐degron pathways and to unveil how normal proteomes are exempt from C‐degron pathway‐mediated destruction. Our data reveal that C‐degron pathways remove mislocalized cellular proteins and cleavage products of deubiquitinating enzymes. Furthermore, the C‐degron and N‐degron pathways cooperate in protein removal. Proteome analysis revealed a shortfall in normal proteins targeted by C‐degron pathways, but not of defective proteins, suggesting proteolysis‐based immunity as a constraint for protein evolution/selection. Our work highlights the importance of protein termini for protein quality surveillance, and the relationship between the functional proteome and protein degradation pathways.     

Molecular interactions of SHP1 and SHP2 in IL-3-signalling.

SHP1 and SHP2 tyrosine phosphatases have both been implicated in signalling pathways downstream of the interleukin-3 (IL-3) receptor. We have investigated the co-association of SHP1 and SHP2 with tyrosine-phosphorylated proteins in IL-3-dependent BaF/3 cells. We demonstrate that both SHP1 and SHP2 associate with Aic2A (beta chain of the IL-3 receptor), Gab2 and the paired inhibitory receptor B (PIR-B). The individual SH2 domains of SHP2 can independently bind Gab2, potentially important for the adapter function of SHP2. Association of both phosphatases with Aic2A and Gab2 increases upon IL-3 treatment. Recruitment of SHP1 to PIR-B also increases in response to IL-3, suggesting a functional link between inhibitory and cytokine receptor signalling. Aic2A is a rapid target for dephosphorylation following IL-3 stimulation and substrate-trapping versions of both phosphatases identify Aic2A and Gab2 as substrates for SHP1 and SHP2. These studies suggest that SH2-domain interactions are important for targetting these phosphatases to their substrates.     

A novel role for Gab1 and SHP2 in epidermal growth factor-induced Ras activation

SHP2 was recently found to down-regulate PI3K activation by dephosphorylating Gab1 but the mechanisms explaining the positive role of the Gab1/SHP2 pathway in EGF-induced Ras activation remain ill defined. Substrate trapping experiments now suggest that SHP2 dephosphorylates other Gab1 phosphotyrosines located within a central region displaying four YXXP motifs. Because these sites are potential docking motifs for Ras-GAP, we tested whether SHP2 dephosphorylates them to facilitate Ras activation. We observed that a Gab1 construct preventing SHP2 recruitment promoted membrane relocation of RasGAP. Moreover, a RasGAP-inactive mutant restored the activation of Ras in cells transfected with SHP2-inactivating Gab1 mutant or in SHP2-deficient fibroblasts, supporting the hypothesis that RasGAP is a downstream target of SHP2. To determine whether Gab1 is a RasGAP-binding partner, a Gab1 mutant deleted of four YXXP motifs was produced. The deletion suppressed RasGAP redistribution and restored the defective Ras activation caused by SHP2-inactivating mutations. Moreover, Gab1 was found to interact with RasGAP SH2 domains, only under conditions where SHP2 is not activated. To identify Ras-GAP-binding sites, Tyr to Phe mutants of Gab1 YXXP motifs were produced. Gab1 constructs mutated on Tyr(317) were severely affected in RasGAP binding and were the most active in compensating for Ras-defective activation and blocking RasGAP redistribution induced by SHP2 inactivation. We have thus localized on Gab1 a Ras-negative regulatory tyrosine phosphorylation site involved in RasGAP binding and showed that an important SHP2 function is to down-regulate its phosphorylation to disengage RasGAP and sustain Ras activation.     

Gab3, a New DOS/Gab Family Member, Facilitates Macrophage Differentiation

Using the FDC-P1 cell line expressing the exogenous macrophage colony-stimulating factor (M-CSF) receptor, Fms, we have analyzed the role of a new mammalian DOS/Gab-related signaling protein, called Gab3, in macrophage cell development of the mouse. Gab3 contains an amino-terminal pleckstrin homology domain, multiple potential sites for tyrosine phosphorylation and SH2 domain binding, and two major polyproline motifs potentially interacting with SH3 domains. Among the growing family of Gab proteins, Gab3 exhibits a unique and overlapping pattern of expression in tissues of the mouse compared with Gab1 and Gab2. Gab3 is more restricted to the hematopoietic tissues such as spleen and thymus but is detectable at progressively lower levels within heart, kidney, uterus, and brain. Like Gab2, Gab3 is tyrosine phosphorylated after M-CSF receptor stimulation and associates transiently with the SH2 domain-containing proteins p85 and SHP2. Overexpression of exogenous Gab3 in FD-Fms cells dramatically accelerates macrophage differentiation upon M-CSF stimulation. Unlike Gab2, which shows a constant mRNA expression level after M-CSF stimulation, Gab3 expression is initially absent or low in abundance in FD cells expressing the wild-type Fms, but Gab3 mRNA levels are increased upon M-CSF stimulation. Moreover, M-CSF stimulation of FD-FmsY807F cells (which grow but do not differentiate) fails to increase Gab3 expression. These results suggest that Gab3 is important for macrophage differentiation and that differentiation requires the early phosphorylation of Gab2 followed by induction and subsequent phosphorylation of Gab3.     

ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase

We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2.     

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.     

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1

Intrinsically disordered proteins (IDPs) are an important class of proteins which lack tertiary structure elements. Their dynamic properties can depend on reversible post-translational modifications and the complex cellular milieu, which provides a crowded environment. Both influences the thermodynamic stability and folding of globular proteins as well as the conformational plasticity of IDPs. Here we investigate the intrinsically disordered C-terminal region (amino acids 613–694) of human Grb2-associated binding protein 1 (Gab1), which binds to the disease-relevant Src homolog region 2 (SH2) domain-containing protein tyrosine phosphatase SHP2 (PTPN11). This binding is mediated by phosphorylation at Tyr 627 and Tyr 659 in Gab1. We characterize induced structure in Gab1_613–694 and binding to SHP2 by NMR, CD and ITC under non-crowding and crowding conditions, employing chemical and biological crowding agents and compare the results of the non-phosphorylated and tyrosine phosphorylated C-terminal Gab1 fragment. Our results show that under crowding conditions pre-structured motifs in two distinct regions of Gab1 are formed whereas phosphorylation has no impact on the dynamics and IDP character. These structured regions are identical to the binding regions towards SHP2. Therefore, biological crowders could induce some SHP2 binding capacity. Our results therefore indicate that high concentrations of macromolecules stabilize the preformed or excited binding state in the C-terminal Gab1 region and foster the binding to the SH2 tandem motif of SHP2, even in the absence of tyrosine phosphorylation.     

TSGIT: An N‐ and C‐terminal tandem tag system for purification of native and intein‐mediated ligation‐ready proteins

A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.     

A yeast two-hybrid study of human p97/Gab2 interactions with its SH2 domain-containing binding partners

p97/Gab2 is a recently characterized member of a large family of scaffold proteins that play essential roles in signal transduction. Gab2 becomes tyrosine-phosphorylated in response to a variety of growth factors and forms multimolecular complexes with SH2 domain-containing signaling molecules such as the p85-regulatory subunit of the phosphoinositide-3-kinase (p85-PI3K), the tyrosine phosphatase SHP-2 and the adapter protein CrkL. To characterize the interactions between Gab2 and its SH2-containing binding partners, we designed a modified yeast two-hybrid system in which the Lyn tyrosine kinase is expressed in a regulated manner in yeast. Using this assay, we demonstrated that p97/Gab2 specifically interacts with the SH2 domains of PI3K, SHP-2 and CrkL. Interaction with p85-PI3K is mediated by tyrosine residues Y452, Y476 and Y584 of Gab2, while interaction with SHP-2 depends exclusively on tyrosine Y614. CrkL interaction is mediated by its SH2 domain recognizing Y266 and Y293, despite the latter being in a non-consensus (YTFK) environment.     

Previously uncharacterized interactions between the folded and intrinsically disordered domains impart asymmetric effects on UBQLN2 phase separation

Shuttle protein UBQLN2 functions in protein quality control (PQC) by binding to proteasomal receptors and ubiquitinated substrates via its N‐terminal ubiquitin‐like (UBL) and C‐terminal ubiquitin‐associated (UBA) domains, respectively. Between these two folded domains are low‐complexity STI1‐I and STI1‐II regions, connected by disordered linkers. The STI1 regions bind other components, such as HSP70, that are important to the PQC functions of UBQLN2. We recently determined that the STI1‐II region enables UBQLN2 to undergo liquid–liquid phase separation (LLPS) to form liquid droplets in vitro and biomolecular condensates in cells. However, how the interplay between the folded (UBL/UBA) domains and the intrinsically disordered regions mediates phase separation is largely unknown. Using engineered domain deletion constructs, we found that removing the UBA domain inhibits UBQLN2 LLPS while removing the UBL domain enhances LLPS, suggesting that UBA and UBL domains contribute asymmetrically in modulating UBQLN2 LLPS. To explain these differential effects, we interrogated the interactions that involve the UBA and UBL domains across the entire UBQLN2 molecule using nuclear magnetic resonance spectroscopy. To our surprise, aside from well‐studied canonical UBL:UBA interactions, there also exist moderate interactions between the UBL and several disordered regions, including STI1‐I and residues 555–570, the latter of which is a known contributor to UBQLN2 LLPS. Our findings are essential for the understanding of both the molecular driving forces of UBQLN2 LLPS and the effects of ligand binding to UBL, UBA, or disordered regions on the phase behavior and physiological functions of UBQLN2.     

Calcium Release at Fertilization in Starfish Eggs Is Mediated by Phospholipase Cγ

Although inositol trisphosphate (IP₃) functions in releasing Ca²⁺ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP₃. To distinguish between a role for PLCγ, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCβ, which is activated by a G protein, we injected starfish eggs with a PLCγ SH2 domain fusion protein that inhibits activation of PLCγ. In these eggs, Ca²⁺ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCγSH2 protein is a specific inhibitor of PLCγ in the egg, since it did not inhibit PLCβ activation of Ca²⁺ release initiated by the serotonin 2c receptor, or activation of Ca²⁺ release by IP₃ injection. Furthermore, injection of a PLCγ SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca²⁺ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cγ by an SH2 domain-mediated process stimulates the production of IP₃ that causes intracellular Ca²⁺ release.