TMEM43/LUMA is a key signaling component mediating EGFR-induced NF-κB activation and tumor progression.

Epidermal growth factor receptor (EGFR) family members play pivotal roles in cell proliferation, differentiation and survival. Overexpression and mutations of EGFRs, or aberrant EGFR signaling are commonly associated with the development of various cancers, where constitutive NF-κB activation is often found to promote the expression of various proteins involved in the proliferation, survival, migration and epithelial-to-mesenchymal transition of cancer cells. However, the mechanism of EGFR-induced NF-κB activation is not fully defined. Here, we used a Bimolecular Fluorescence Complementation-based functional genomics method to perform a high throughput screening and identified TMEM43/LUMA as a critical component in EGFR signaling network, mediating EGFR-induced NF-κB activation. Our data show that EGFR recruits TMEM43 following EGF stimulation. TMEM43 interacts with the scaffold protein CARMA3 and its associating complex to induce downstream NF-κB activation, and plays a critical role in controlling cell survival. TMEM43 deficiency significantly affects colony formation, survival of anoikis-induced cell death, migration and invasion of cancer cells in vitro, as well as tumor progression in vivo. Importantly, higher expression of TMEM43 closely correlates with brain tumor malignancy, and suppression of TMEM43 expression in brain tumor cells inhibited their growth both in vitro and in vivo. Altogether, our studies reveal a crucial link of EGF receptor to NF-κB activation and tumor progression.

CHMP6 and VPS4A mediate the recycling of Ras to the plasma membrane to promote growth factor signaling.

While Ras is well-known to function on the plasma membrane (PM) to mediate growth factor signaling, increasing evidence suggests that Ras has complex roles in the cytoplasm. To uncover these roles, we screened a cDNA library and isolated H-Ras-binding proteins that also influence Ras functions. Many isolated proteins regulate trafficking involving endosomes; CHMP6/VPS20 and VPS4A, which interact with ESCRT-III (Endosomal Sorting Complex Required for Transport-III), were chosen for further study. We showed that the binding is direct and occurs in endosomes. Furthermore, the binding is most efficient when H-Ras has a functional effector-binding loop, and is GTP-bound and ubiquitylated. CHMP6 and VPS4A also bound to N-Ras but not K-Ras. Repressing CHMP6 and VPS4A blocked Ras-induced transformation, which correlated with inefficient Ras localization to the PM as measured by cell fractionation and photobleaching. Moreover, silencing CHMP6 and VPS4A also blocked epidermal growth factor receptor (EGFR) recycling. These data suggest that Ras interacts with key ESCRT-III components to promote recycling of itself and EGFR back to the PM to create a positive feedback loop to enhance growth factor signaling.

Regulation of cytokine-independent survival kinase (CISK) by the Phox homology domain and phosphoinositides.

PKB/Akt and serum and glucocorticoid-regulated kinase (SGK) family kinases are important downstream targets of phosphatidylinositol 3 (PI-3) kinase and have been shown to mediate a variety of cellular processes, including cell growth and survival. Although regulation of Akt can be achieved through several mechanisms, including its phosphoinositide-binding Pleckstrin homology (PH) domain, how SGK kinases are targeted and regulated remains to be elucidated. Unlike Akt, cytokine-independent survival kinase (CISK)/SGK3 contains a Phox homology (PX) domain. PX domains have been implicated in several cellular events involving membrane trafficking. However, their precise function remains unknown. We demonstrate here that the PX domain of CISK interacts with phosphatidylinositol (PtdIns)(3,5)P2, PtdIns(3,4,5)P3, and to a lesser extent PtdIns(4,5)P2. The CISK PX domain is required for targeting CISK to the endosomal compartment. Mutation in the PX domain that abolished its phospholipid binding ability not only disrupted CISK localization, but also resulted in a decrease in CISK activity in vivo. These results suggest that the PX domain regulates CISK localization and function through its direct interaction with phosphoinositides. Therefore, CISK and Akt have evolved to utilize different lipid binding domains to accomplish a similar mechanism of activation in response to PI-3 kinase signaling.

Domain-dependent function of the rasGAP-binding protein p62Dok in cell signaling.

p62Dok, the rasGAP-binding protein, is a common target of protein-tyrosine kinases. It is one of the major tyrosine-phosphorylated molecules in v-Src-transformed cells. Dok consists of an amino-terminal Pleckstrin homology domain, a putative phosphotyrosine binding domain, and a carboxyl-terminal tail containing multiple tyrosine phosphorylation sites. The importance and function of these sequences in Dok signaling remain largely unknown. We have demonstrated here that the expression of Dok can inhibit cellular transformation by the Src tyrosine kinase. Both the phosphotyrosine binding domain and the carboxyl-terminal tail of Dok (in particular residues 336-363) are necessary for such activity. Using a combinatorial peptide library approach, we have shown that the Dok phosphotyrosine binding domain binds phosphopeptides with the consensus motif of Y/MXXNXL-phosphotyrosine. Furthermore, Dok can homodimerize through its phosphotyrosine binding domain and Tyr(146) at the amino-terminal region. Mutations of this domain or Tyr(146) that block homodimerization significantly reduce the ability of Dok to inhibit Src transformation. Our results suggest that Dok oligomerization through its multiple domains plays a critical role in Dok signaling in response to tyrosine kinase activation.

A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase.

We utilized a novel peptide library approach to identify specific inhibitors of ZAP-70, a protein Tyr kinase involved in T cell activation. By screening more than 6 billion peptides oriented by a common Tyr residue for their ability to bind to ZAP-70, we determined a consensus optimal peptide. A Phe-for-Tyr substituted version of the peptide inhibited ZAP-70 protein Tyr kinase activity by competing with protein substrates (K(I) of 2 microM). The related protein Tyr kinases, Lck and Syk, were not significantly inhibited by the peptide. When introduced into intact T cells, the peptide blocked signaling downstream of ZAP-70, including ZAP-70-dependent gene induction, without affecting upstream Tyr phosphorylation. Thus, screening Tyr-oriented peptide libraries can identify selective peptide inhibitors of protein Tyr kinases.

Identification of CISK, a new member of the SGK kinase family that promotes IL-3-dependent survival.

The signaling pathways for cell survival are much less well understood than those for apoptosis [1]. Many mammalian cell-survival factors have been identified, either biochemically or from genetic studies in other organisms. Effective genetic methods that allow systematic study of anti-apoptosis genes in mammalian cells remain to be established, however. To achieve this goal, we used a new genetic screening method using enhanced retroviral mutagen (ERM) vectors to identify factors that mediate IL-3-dependent survival of hematopoietic cells. Both known and novel mediators of cell survival were identified, including Bcl-xL, phosphatidylinositide 3-kinase (PI 3-kinase), Akt and cytokine-independent survival kinase (CISK). CISK is a PX-domain-containing serine/threonine kinase homologous to serum- and glucocorticoid-regulated protein kinase (SGK). We showed that CISK acts downstream of the PI 3-kinase cascade in vivo and may function in parallel to Akt by phosphorylating Bad and the transcription factor FKHRL1. The distinct subcellular localization of CISK, however, suggests that it acts in different signaling cascades from Akt. Our results demonstrate the power of ERM to identify key genes involved in cell-survival signaling. Furthermore, CISK is the first SGK family member that has been shown to promote survival, pointing to the possibility that other SGK family proteins may also function in survival pathways.

Why does threonine, and not serine, function as the active site nucleophile in proteasomes?

Proteasomes belong to the N-terminal nucleophile group of amidases and function through a novel proteolytic mechanism, in which the hydroxyl group of the N-terminal threonines is the catalytic nucleophile. However, it is unclear why threonine has been conserved in all proteasomal active sites, because its replacement by a serine in proteasomes from the archaeon Thermoplasma acidophilum (T1S mutant) does not alter the rates of hydrolysis of Suc-LLVY-amc (Seemüller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. (1995) Science 268, 579-582) and other standard peptide amide substrates. However, we found that true peptide bonds in decapeptide libraries were cleaved by the T1S mutant 10-fold slower than by wild type (wt) proteasomes. In degrading proteins, the T1S proteasome was 3.5- to 6-fold slower than the wt, and this difference increased when proteolysis was stimulated using the proteasome-activating nucleotidase (PAN) ATPase complex. With mutant proteasomes, peptide bond cleavage appeared to be rate-limiting in protein breakdown, unlike with wt. Surprisingly, a peptide ester was hydrolyzed by both particles much faster than the corresponding amide, and the T1S mutant cleaved it faster than the wt. Moreover, the T1S mutant was inactivated by the ester inhibitor clasto-lactacystin-beta-lactone severalfold faster than the wt, but reacted with nonester irreversible inhibitors at similar rates. T1A and T1C mutants were completely inactive in all these assays. Thus, proteasomes lack additional active sites, and the N-terminal threonine evolved because it allows more efficient protein breakdown than serine.

Genetic screens in mammalian cells by enhanced retroviral mutagens.

Genetic approaches such as retrovirus-mediated mutagenesis and cDNA expression libraries have contributed greatly to our understanding of signal transduction in mammalian cells. However, previously described methods for retroviral insertional mutagenesis are hindered by low mutagenesis rates and difficulties in cloning mutated genes. cDNA expression library methods are usually cell-type dependent and bias towards abundant and short messages. With the near completion of the genome projects, alternative genetic methods are needed where large numbers of genes can be more easily isolated and biochemically studied. We have developed a novel retrovirus-mediated genetic screening method in cultured cells. To achieve efficient and regulated mutagenesis, we constructed Enhanced Retroviral Mutagen (ERM) vectors that contained several engineered sequences (e.g., an ERM Tag and a splice donor) controlled by a tetracycline-responsive promoter. Endogenous genes can thus be randomly activated and tagged in a conditional system. NIH3T3 cells were used to screen for focus-forming genes using the ERM strategy. We showed that these added sequences increased the screening efficiency by >10-fold, and allowed more direct identification of the genes targeted. Sequence analysis of approximately 10% of the >600 focus clones recovered revealed both known oncogenes and novel factors such as protein kinases and GTP/GDP exchange proteins. The ERM strategy should help to facilitate large-scale gene identification in diverse pathways and integrate both genetic (with the completion of the genome projects) and functional information more readily.

The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif.

Ephrin B proteins function as ligands for B class Eph receptor tyrosine kinases and are postulated to possess an intrinsic signaling function. The sequence at the carboxyl terminus of B-type ephrins contains a putative PDZ binding site, providing a possible mechanism through which transmembrane ephrins might interact with cytoplasmic proteins. To test this notion, a day 10.5 mouse embryonic expression library was screened with a biotinylated peptide corresponding to the carboxyl terminus of ephrin B3. Three of the positive cDNAs encoded polypeptides with multiple PDZ domains, representing fragments of the molecule GRIP, the protein syntenin, and PHIP, a novel PDZ domain-containing protein related to Caenorhabditis elegans PAR-3. In addition, the binding specificities of PDZ domains previously predicted by an oriented library approach (Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., and Cantley, L. C. (1997) Science 275, 73-77) identified the tyrosine phosphatase FAP-1 as a potential binding partner for B ephrins. In vitro studies demonstrated that the fifth PDZ domain of FAP-1 and full-length syntenin bound ephrin B1 via the carboxyl-terminal motif. Lastly, syntenin and ephrin B1 could be co-immunoprecipitated from transfected COS-1 cells, suggesting that PDZ domain binding of B ephrins can occur in cells. These results indicate that the carboxyl-terminal motif of B ephrins provides a binding site for specific PDZ domain-containing proteins, which might localize the transmembrane ligands for interactions with Eph receptors or participate in signaling within ephrin B-expressing cells.

Stimulation through the T cell receptor leads to interactions between SHB and several signaling proteins.

Shb is a recently described Src homology 2 (SH2) domain-containing adaptor protein. Here we show that Shb is expressed in lymphoid tissues, and is recruited into signaling complexes upon activation of Jurkat T cells. Grb2 binds proline-rich motifs in Shb via its SH3 domains. As a result, a number of proteins detected in anti-Shb and anti-Grb2 immunoprecipitates are shared, including phosphoproteins of 22, 36/38, 55/57 and 70 kDa. Shb-association with p22, which represents the T cell receptor associated zeta chain, occurs through the Shb SH2 domain. The central region of Shb binds p36/38. Since this interaction was inhibited by phosphotyrosine, this region of Shb is likely to contain a non-SH2 PTB (phosphotyrosine binding) domain. The Shb PTB domain was found to preferentially bind the sequence Asp-Asp-X-pTyr when incubated with a phosphopeptide library. A peptide corresponding to a phosphorylation site in 34 kDa Lnk inhibited association between Shb and p36/38. Overexpression of Shb in Jurkat cells led to increased basal phosphorylation of Shb-associated p36/38 and p70 proteins. Inactivation of the Shb SH2 domain by an R522K mutation resulted in a reduced stimulation of tyrosine phosphorylation of several proteins in response to CD3 crosslinking when expressed in Jurkat cells. Together, our results show three distinct domains of Shb all participate in the formulation of multimeric signaling complexes in activated T cells. These results indicate that the Shb protein functions in T cell receptor signaling.

SRPK2: a differentially expressed SR protein-specific kinase involved in mediating the interaction and localization of pre-mRNA splicing factors in mammalian cells.

Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain-containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain-containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

Peptide and protein phosphorylation by protein tyrosine kinase Csk: insights into specificity and mechanism.

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in down-regulation of Src family members. The molecular basis of Csk's substrate specificity and catalytic mechanism with a protein substrate was investigated. Using a peptide library approach, preferential amino acids which are unrelated to the conserved Src C-terminal sequence were identified. The validity of these preferences was confirmed by synthesizing a short consensus peptide and demonstrating its high catalytic efficiency with Csk. These results underscore the difficulties of relying on amino acids neighboring tyrosine in protein sequences as predictors of protein kinase substrate specificity for in vivo analysis. In addition, a catalytically inactive version of the Src family member, Lck (lymphoid cell kinase), was expressed, purified, and evaluated as a Csk substrate. It was proven to be the most catalytically efficient substrate yet identified for Csk. The high efficiency of purified Csk phosphorylating a pure, unphosphorylated Src family member argues against the importance of an SH2-phosphotyrosine docking interaction or the involvement of extra recruitment proteins in facilitating Csk phosphorylation of Src family members. Kinetic studies revealed that the chemical step is at least partially rate-determining in Csk-mediated phosphoryl transfer to the Lck protein. Other properties including preferences for Mn over Mg, thio effects, and Km's for ATP also correlate fairly well between protein and peptide phosphorylation. The lack of a significant impact of increased salt on the Km for Lck phosphorylation differs from Csk-mediated poly(Glu,Tyr) phosphorylation, and argues against the importance of electrostatic effects in the Csk-Lck binding interaction. The failure of the Lck phosphorylation product (phosphotyrosine-505) to significantly inhibit Csk phosphorylation of Lck is consistent with a catalytic model involving multidomain structural interactions between substrate and enzyme.

Interleukin 3-dependent survival by the Akt protein kinase.

Interleukin 3 (IL-3)-dependent survival of hematopoietic cells is known to rely on the activity of multiple signaling pathways, including a pathway leading to activation of phosphoinositide 3-kinase (PI 3-kinase), and protein kinase Akt is a direct target of PI 3-kinase. We find that Akt kinase activity is rapidly induced by the cytokine IL-3, suggesting a role for Akt in PI 3-kinase-dependent signaling in hematopoetic cells. Dominant-negative mutants of Akt specifically block Akt activation by IL-3 and interfere with IL-3-dependent proliferation. Overexpression of Akt or oncogenic v-akt protects 32D cells from apoptosis induced by IL-3 withdrawal. Apoptosis after IL-3 withdrawal is accelerated by expression of dominant-negative mutants of Akt, indicating that a functional Akt signaling pathway is necessary for cell survival mediated by the cytokine IL-3. Thus Akt appears to be an important mediator of anti-apoptotic signaling in this system.

Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells.

Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, we have expressed the EphB2 receptor (formerly Nuk/Cek5/Sek3) in neuronal NG108-15 cells, and have observed the tyrosine phosphorylation of multiple cellular proteins upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induced the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn formed a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also bound through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexed with activated EphB2 in vivo. We have localized an in vitro RasGAP-binding site to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2, and demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signalling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the + 3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signalling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture.

High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif.

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYphi motif (phi represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 +/- 0.74 microM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 +/- 0.10 microM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.

Sequence specificity of C-terminal Src kinase (CSK)--a comparison with Src-related kinases c-Fgr and Lyn.

An eicosapeptide encompassing the C-terminal tail of c-Src (Tyr527) which is conserved in most Src-related protein kinases, is phosphorylated by C-terminal Src kinase (CSK) and by the two Src-related protein kinases c-Fgr and Lyn, with similar kinetic constants. Two related peptides reproducing the C-terminal segments of c-Src mutants defective in CSK phosphorylation [MacAuley, A., Okada, M., Nada, S., Nakagawa, H. & Cooper, J. A. (1993) Oncogene 8, 117-124] AFLEDSCTGTEPLYQRGENL (mutant number 28) and AFLEDNFTGTKPQYHPGENL (mutant number 29), proved a better and a much worse substrates, respectively than the wild-type peptide, with either CSK or the two Src kinases. By changing individual residues in the best peptide substrate, it was shown that the main element responsible for its improved phosphorylation is leucine at position -1 (instead of glutamine), while lysine at position -3 (instead of glutamate) has a detrimental effect, possibly accounting for the negligible phosphorylation of peptide derived from mutant number 29. By contrast to most peptide substrates, including the Src C-terminal peptides, which exhibit relatively high K(m) values, a polyoma-virus-middle-T-antigen-(mT)-derived peptide with tyrosine embedded in a highly hydrophobic sequence (EEEPQFEEIPIYLELLP) exhibits with CSK a quite low K(m) value (63 microM). Consistent with this, the optimal sequence selected by CSK in an oriented peptide library is XXXIYMFFF. This is different from sequences selected by Lyn (DEEIYEELX) and c-Fgr (XEEIYGIFF), although they all share a high selection for a hydrophobic residue at n-1. In sharp contrast, TPKIIB/p38syk, related to the catalytic domain of p72syk, selects acidic residues at nearly all positions, n-1 included. These data support the notion that the features determining the specific phosphorylation of the C-terminal tyrosine residue of Src do not reside in the primary structure surrounding the target tyrosine. They also show that this site does not entirely fulfil the optimal consensus sequence recognized by CSK, disclosing the possibility that as yet unrecognized CSK targets structurally unrelated to the C-terminal tyrosine residue of Src kinases may exist.