Stephan M Feller’s research while affiliated with Martin Luther University Halle-Wittenberg and other places

What is this page?


This page lists works of an author who doesn't have a ResearchGate profile or hasn't added the works to their profile yet. It is automatically generated from public (personal) data to further our legitimate goal of comprehensive and accurate scientific recordkeeping. If you are this author and want this page removed, please let us know.

Publications (199)


Figure 1. SHP2 structural elements. (A) Domain organisation of SHP2 (human residue numbering), consisting of two SH2 domains (blue and red) joined by a short linker (green) followed by the protein tyrosine phosphatase (PTP) catalytic domain (yellow). The latter possesses a C-terminal extension that can be dispensed with for activity measurements and is truncated in SHP2 structures. (B) Crystal structure of SHP2 in its autoinhibited state (Hof et al. 1998) (pdb code 2shp). The N-terminal SH2 domain blocks access to the PTP active site, marked by cyan spheres showing the phosphate position of phospho-tyrosine (pY) products. To aid orientation, locations of the SH2 pY phosphate binding sites are marked by green spheres and SH2 B helices (see Figure 2) are in separate tones (B N-SH2 light violet, B N-SH2 brown). (C) The constitutively active oncogenic SHP2 E76K mutation E76K results in a substantial reorganisation of the N- and C-SH2 domains (LaRochelle et al. 2018) (pdb 6crf), unmasking the active site for substrate phosphorylation. Orientation of the PTP domain as in (B). (D) Sequence of Gab1 611-694 , with (p)Y residues (p)Y 627 and (p)Y 659 marked in green.
Figure 3. Structures of the SHP2 1-222 tandem domain in the presence and absence of activator peptides, aligned through superposition of the N-SH2 domains (blue). (A) The tandem N-SH2-C-SH2 domain SHP2 1-220 in the presence of bis-phosphorylated pY 627 pY 659 -Gab1 617-683 (green; disordered residues Leu 632 -Val 652 represented by a dotted line) solved by microED (this work). The crystal structure reveals a novel inter-domain interface (B) centred around Pro 144 that has previously not been observed for SHP2. (C) SH2 domain arrangement in autoinhibited SHP2 (Hof et al. 1998), ie in the absence of any activator. Nearly all deposited SHP2 structures show this arrangement (not shown). (D) Relative orientations of the SH2 domains in the constitutively active oncogenic mutant SHP2 E76K (LaRochelle et al. 2018) without activator. (E-G) SHP2 tandem domains in complex with monophosphorylated peptides from Helicobacter pylori CagA (Hayashi et al. 2017) (E, F) or thioredoxin-interacting protein TXNIP (G). (H, I) An interface corresponding to that seen here (A, B) is found in an open crystal structure of the related SHP1/PTPN6 enzyme (Wang et al. 2011)). For orientation, SHP1 helices B N-SH2 and B N-SH2 are coloured cyan and pink respectively. (J) Selected regions of the 2D-TROSY spectra [Figure S7] from 15 N-labelled SHP2 1-222 (black) in the presence of pY 627 pY 659 -Gab1 613 -694 superimposed on those of the peptide bound 15 N-N-SH2 and 15 N-C-SH2 domains (both red).
Figure 4. (A) Model of the postulated pYpY-Gab1 activated state of SHP2. (B) Superposition of the two phosphotyrosine peptides to the respective N- and C-SH2 domains in the autoinhibited structure requires an almost fully extended conformation to bridge by the intervening 26 residues, which would allow simultaneous binding to both SH2 domains in this conformation. (C) Postulated mechanism for the activation of SHP2 by bisphosphorylated Gab1. Binding of pY 627 destabilises the N-SH2 domain, resulting in its dissociation from the PTP domain and activation for the phosphotyrosine phosphatase. The C-SH2 domain relocates to its favoured position on the catalytic domain, engaging pY 659 , with the N-SH2 following. The multiple small energetic contributions to each state would allow for rapid conversion between inactive and active states that could be additionally modulated through e.g. dephosphorylation of the Gab1 peptide.
Mechanism of SHP2 activation by bis-Tyr-phosphorylated Gab1
  • Preprint
  • File available

December 2024

·

28 Reads

·

·

Tobias Gruber

·

[...]

·

The non-receptor tyrosine phosphatase SHP2 (PTPN11) is a regulator of diverse cellular functions including mitogenic activation and cell migration. SHP2 consists of two tandem SH2 domains followed by the catalytic domain, and is autoinhibited by the N-terminal SH2 domain that blocks access to the active site. Mutations that influence auto-inhibition have been implicated in cancer and other diseases, and allosteric inhibitors have been developed that stabilise the inactive state. The mechanism of SHP2 activation remains unclear, however. Here, we show that the intrinsically disordered bis-phosphorylated SHP2-activating peptide pY627pY659-Gab1 binds to both SH2 domains, undergoing a partial disorder-to-order transition in the process. In addition to eliciting changes in SH2 domain dynamics, the peptide reorganises their relative orientations to provide a new SH2-SH2 interface. Our data suggest an active conformation for SHP2 that is also applicable to the hematopoietic cell-specific SHP1 (PTPN6), shedding light on the activation mechanism of both enzymes and paving the way for the development of novel compounds that modulate SHP2 activity.

Download

The Src family kinase inhibitor drug Dasatinib and glucocorticoids display synergistic activity against tongue squamous cell carcinoma and reduce MET kinase activity

October 2024

·

11 Reads

Background: Tongue squamous cell carcinoma (TSCC) is an aggressive cancer associated with a poor prognosis and limited treatment options, necessitating new drug targets to improve therapeutic outcomes. Our current work focuses on protein tyrosine kinases as well-known targets for successful cancer therapies. Methods: Western blot analysis of tyrosine phosphorylation patterns in 34 TSCC lines identified Src family kinases (SFKs) as the main contributors. Inhibition of SFKs with PP2 and Dasatinib led to profound biological effects. A high-throughput screen with 1600 FDA-approved drugs was performed with three TSCC lines to discover drugs that act synergistically with Dasatinib against TSCC cell viability. Glucocorticoids emerged as potential candidates and were further investigated in 2D culture and by 3D soft agar colony formation. Dexamethasone was chosen as the major tool for our analyses. Since Dasatinib and glucocorticoids are known for their pleiotropic actions on cells, we analyzed effects on cell cycle, senescence, autophagy and cell signaling. Results: A panel of 34 TSCC lines showed a surprisingly homogenous pTyr-protein pattern and a prominent 130 kDa pTyr-protein. Inhibition of SFK activity greatly reduced overall pTyr-protein levels and p130Cas tyrosine phosphorylation. It also impaired TSCC viability in 2D cell culture and 3D soft agar colony formation. A high-throughput drug combination screen with Dasatinib identified glucocorticoids as promising candidates for synergistic activity. Dasatinib and Dexamethasone combination treatment showed strong synergistic effects on Src and p130Cas phosphorylation and led to reduced p130Cas expression. Dexamethasone also suppressed phosphorylation of the MET kinase and its key substrate Gab1. On the cellular level, Dasatinib combination with glucocorticoids led to G1 cell cycle arrest, increased senescence and enhanced autophagy. This was also reflected by effects on cell cycle regulatory proteins, including CDKs and cyclins. Conclusion: This work is the first to show a strong synergistic activity of Dasatinib in combination with clinically used glucocorticoids in solid tumors. Furthermore, the tyrosine kinase MET and its effector protein Gab1 are newly identified glucocorticoid targets. Given the extensive research on MET as a drug target in various cancers, our findings have the potential to advance future cancer treatments. Keywords: Tongue squamous cell carcinoma, combination therapy, targeted therapy, glucocorticoids, Src family kinases.


Structural basis of binding the unique N-terminal domain of microtubule-associated protein 2c to proteins regulating kinases of signaling pathways

July 2024

·

14 Reads

Journal of Biological Chemistry

Isoforms of microtubule-associated protein 2 (MAP2) differ from their homolog Tau in the sequence and interactions of the N-terminal region. Binding of the N-terminal region of MAP2c (N-MAP2c) to the dimerization/docking domains of the regulatory subunit RIIα of cAMP-dependent protein kinase (RIIDD2) and to the Src-homology domain 2 (SH2) of growth factor receptor-bound protein 2 (Grb2) have been described long time ago. However, the structural features of the complexes remained unknown due to the disordered nature of MAP2. Here, we provide structural description of the complexes. We have solved solution structure of N-MAP2c in complex with RIIDD2, confirming formation of an amphiphilic α-helix of MAP2c upon binding, defining orientation of the α-helix in the complex and showing that its binding register differs from previous predictions. Using chemical shift mapping, we characterized the binding interface of SH2-Grb2 and rat MAP2c phosphorylated by the tyrosine kinase Fyn in their complex and proposed a model explaining differences between SH2-Grb2 complexes with rat MAP2c and phosphopeptides with a Grb2-specific sequence. The results provide the structural basis of a potential role of MAP2 in regulating cAMP-dependent phosphorylation cascade via interactions with RIIDD2 and Ras signaling pathway via interactions with SH2-Grb2.


Epstein-Barr virus-driven B cell lymphoma mediated by a direct LMP1-TRAF6 complex

January 2024

·

125 Reads

·

8 Citations

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) drives viral B cell transformation and oncogenesis. LMP1’s transforming activity depends on its C-terminal activation region 2 (CTAR2), which induces NF-κB and JNK by engaging TNF receptor-associated factor 6 (TRAF6). The mechanism of TRAF6 recruitment to LMP1 and its role in LMP1 signalling remains elusive. Here we demonstrate that TRAF6 interacts directly with a viral TRAF6 binding motif within CTAR2. Functional and NMR studies supported by molecular modeling provide insight into the architecture of the LMP1-TRAF6 complex, which differs from that of CD40-TRAF6. The direct recruitment of TRAF6 to LMP1 is essential for NF-κB activation by CTAR2 and the survival of LMP1-driven lymphoma. Disruption of the LMP1-TRAF6 complex by inhibitory peptides interferes with the survival of EBV-transformed B cells. In this work, we identify LMP1-TRAF6 as a critical virus-host interface and validate this interaction as a potential therapeutic target in EBV-associated cancer.


Self-assembly of Grb2 meshworks revealed by Grb2-Gab1 497-528 complex structure

June 2023

·

96 Reads

The ubiquitously expressed adaptor protein Growth factor receptor bound protein 2 (Grb2) plays an essential role in signal transduction by binding to activated receptor tyrosine kinases through its SH2 domain and to downstream effectors via its N- and C-terminal SH3 domains (nSH3, cSH3). Here we present the first structure of ligand-bound full length Grb2. The crystal structure of Grb2 in complex with a bidentate nSH3-cSH3-binding peptide, derived from the multi-site docking protein Grb2-associated binder-1 (Gab1), provides molecular insight into effector recognition by Grb2 and reveals the assembly of a two-dimensional meshwork, consisting of multimeric filament-like Grb2 chains linked to each other by the bivalent bound Gab1 497-528 peptide. Dominant contacts between Grb2 molecules in the multimer are provided by an intermolecular SH2/cSH3 domain interface that is also present in the closed dimer of ligand-free Grb2. We further show that Grb2 is able to self-assemble to form phase-separated condensates in solution. The Grb2 SH2 domain phosphotyrosine binding site is freely accessible in the multimeric assembly, and phase separation is fostered by addition of Gab1 497-528 , as expected from the crystal structure. Multimeric assembly is also observed using a Grb2 SH2-cSH3 didomain construct, and suppressed using a Grb2 Tyr60Glu mutant, a mimic of the in vivo phosphorylated Tyr160 central to the SH2/cSH3 interface, demonstrating that an intact SH2/cSH3 interface is needed for Grb2 assembly in solution.




Values of τ 1/2 (in hours) for phosphorylation of individual MAP2c residues by ERK2 and CDK2 . a Determined for the MAP2c fragment 1-159, b estimated for the MAP2c fragment 250- 347, c determined for the MAP2c fragment 300-467.
Specific phosphorylation of microtubule-associated protein 2c by extracellular signal-regulated kinase reduces interactions at its Pro-rich regions

August 2022

·

42 Reads

·

5 Citations

Journal of Biological Chemistry

Microtubule-associated protein 2 (MAP2) is an important neuronal target of extracellular signal-regulated kinase 2 (ERK2) involved in Raf signaling pathways, but mechanistic details of MAP2 phosphorylation are unclear. Here, we used NMR spectroscopy to quantitatively describe the kinetics of phosphorylation of individual serines and threonines in the embryonic MAP2 variant MAP2c. We carried out real-time monitoring of phosphorylation to discover major phosphorylation sites that were not identified in previous studies relying on specific antibodies. Our comparison with phosphorylation of MAP2c by a model cyclin-dependent kinase CDK2 and with phosphorylation of the MAP2c homolog Tau revealed differences in phosphorylation profiles that explain specificity of regulation of biological functions of MAP2c and Tau. To probe the molecular basis of the regulatory effect of ERK2, we investigated the interactions of phosphorylated and unphosphorylated MAP2c by NMR with single-residue resolution. As ERK2 phosphorylates mostly outside the regions binding microtubules, we studied the binding of proteins other than tubulin, namely regulatory subunit RIIα of cAMP-dependent protein kinase (PKA), adaptor protein Grb2, Src homology domain 3 of tyrosine kinases Fyn and Abl, and ERK2 itself. We found ERK2 phosphorylation interfered mostly with binding to proline-rich regions of MAP2c. Furthermore, our NMR experiments in SH-SY5Y neuroblastoma cell lysates showed that the kinetics of dephosphorylation are compatible with in-cell NMR studies and that residues targeted by ERK2 and PKA are efficiently phosphorylated in the cell lysates. Taken together, our results provide a deeper characterization of MAP2c phosphorylation and its effects on interactions with other proteins.


Epstein-Barr virus-driven B cell lymphoma mediated by a unique LMP1-TRAF6 complex

January 2022

·

58 Reads

·

1 Citation

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) drives viral B cell transformation and oncogenesis. LMP1's transforming activity depends on its cytoplasmic C-terminal activation region 2 (CTAR2), which induces NF-κB and JNK by engaging TNF receptor-associated factor 6 (TRAF6). The mechanism of TRAF6 interaction with LMP1 and its critical role for LMP1 signaling has remained elusive. Here we demonstrate that TRAF6 interacts directly with a novel viral TRAF6 binding motif within CTAR2. Structural modeling supported by NMR and functional studies provides insight into the molecular architecture of the LMP1-TRAF6 complex and reveals substantial differences to CD40-TRAF6 interaction. The direct recruitment of TRAF6 to LMP1 is essential for NF-κB activation and survival of LMP1-driven B cell lymphoma. Disruption of the LMP1-TRAF6 complex by inhibitory peptides interferes with proliferation of EBV-transformed B cells. We identify LMP1-TRAF6 as critical virus-host interface and validate this interaction as novel therapeutic target against EBV.


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

December 2021

·

37 Reads

·

16 Citations

Journal of Molecular Biology

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 Gab1613-694 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.


Citations (41)


... 204-208) is vital for LMP1's interaction with TRAFs, but no specific inhibitor has been developed yet [87]. Similarly, in CTAR2, the amino acid sequence 371-386 [88], particularly the YYD motif (a.a. 384-386), is essential for LMP1's full signaling capacity and represents another promising therapeutic target [87]. ...

Reference:

Epstein-Barr virus-infected nasopharyngeal carcinoma therapeutics: oncoprotein targets and clinical implications
Epstein-Barr virus-driven B cell lymphoma mediated by a direct LMP1-TRAF6 complex

... Although we were unable to obtain crystals of the C-SH2 domain, either in the presence or absence of peptide, the N-SH2-C-SH2-tandem SHP2 1-222 construct in complex with pY 627 pY 659 -Gab1 617-684 yielded long (400 µm), thin crystals with multiple lattices that proved unsuitable for X-ray crystallography. Using 3D electron diffraction (Shaikhqasem et al. 2022), however, orthorhombic microcrystals of the complex could be analysed to yield a structure to 3.2 Å resolution. Interpretable Coulomb potential density is observed for the tandem SH2 domain from R 4 to I 221 (with a break between T 153 and K 164 , i.e. ...

Structure determination of a protein–peptide complex using microcrystal electron diffraction
  • Citing Article
  • August 2022

... During the early stages of neur onal dev elopment, M AP-2 expr ession is low and gradually increases as neurons mature. MAP-2 regulates intrac ellular sig naling pa thw ays by in teracting with other proteins, such as protein kinases, thereby influencing neuronal functions [ 43 ]. Synapses are key structures for communication between neurons, and MAP-2 is inv olv ed in synapse formation and information transmission. ...

Specific phosphorylation of microtubule-associated protein 2c by extracellular signal-regulated kinase reduces interactions at its Pro-rich regions

Journal of Biological Chemistry

... As a result of RANKL binding, RANK activates the expression of TRAF6, which keys to the differentiation of OCPs into osteoclasts. TRAF6 decoy peptides attenuate the differentiation and development of osteoclasts [50]. Our study revealed that AR281 reversed the number of osteoclast-like multinucleated cells and TRAP activity induced by RANKL stimulation. ...

Epstein-Barr virus-driven B cell lymphoma mediated by a unique LMP1-TRAF6 complex

... existing drug resistance (Yi et al. 2023). The formation of binding-competent transient structures induced by molecular crowding in the close vicinity of IDPs/IDRs is another unique mechanism that demonstrates binding promiscuity and multifunctionality (Gruber et al. 2022). These results can potentially lead us to decipher and understand the mechanism of assembly of very large and distinct signal transduction protein complexes (viz., 'signalosomes' that are stimulus specific) in response to certain stimuli in a short period of time (Simister et al. 2011). ...

Macromolecular Crowding Induces a Binding Competent Transient Structure in Intrinsically Disordered Gab1
  • Citing Article
  • December 2021

Journal of Molecular Biology

... Specifically, mention should be made of the Asp165, Ser166, Asp168, and Ser/Leu170 residues. Substitutions D165A, S166A, and D168A result in a lower trimer stability under conditions of polyacrylamide electrophoresis; however, the proteolytic activity of the mutant variants is maintained [76]. ponent of a long loop (aa 153-173), which connects the N-and C-terminal barrels of the proteolytic domain (see the scheme in Figure 3). ...

A novel FRET peptide assay reveals efficient Helicobacter pylori HtrA inhibition through zinc and copper binding

... GRB2 Associated Binding Protein (Gab) family proteins are multisite docking proteins involved in the composition of GRB2. It is engaged in cytokine-induced signal transduction by regulating the recruitment of proteins involved in MAPK/ ERK and PI3K signaling [40]. It has been shown that Gab1 promotes ERK activation and participates in IL-22-mediated proliferation, migration, and differentiation of KCs [41]. ...

The multi-site docking protein Grb2-associated binder 1 (Gab1) enhances interleukin-6-induced MAPK-pathway activation in an SHP2-, Grb2-, and time-dependent manner

Cell Communication and Signaling

... For instance, studies utilizing TCGA data have identified molecular subtypes of CRC, but their prognostic value remains inconsistent in clinical settings [10]. Similarly, CPTAC studies have identified diverse proteomic profiles, yet converting these findings into reliable prognostic markers is challenging [11,12]. This difficulty is primarily due to the vast array of molecular alterations in CRC, complicating the identification of clear prognostic markers [13,14]. ...

Pharmacoproteomic characterisation of human colon and rectal cancer

Molecular Systems Biology

... interaction and consequently allows recruitment of Gab1 to the plasma membrane [23]. Of note, phosphorylation of Gab1 at interaction sites for other proteins occurs both dependent and independent of plasma membrane recruitment and is highly reliant on upstream signalling events [24]. Although a contribution of Gab1 to oncogenic EGFR-induced signalling is known [25,26] the exact role of Gab1 in the MAPK/PI3K signalling network in different TNBC subgroups remains elusive so far. ...

The multi-site docking protein Gab1 is constitutively phosphorylated independent from its recruitment to the plasma membrane in Jak2-V617F-positive cells and mediates proliferation of human erythroleukaemia cells
  • Citing Article
  • March 2017

Cellular Signalling

... Moreover, TEP mRNAs were found be associated with chemotherapeutic effects (74). Together, RNA transcriptome mapping established TEPs as promising biomarker source in liquid biopsies (75)(76)(77). Recently, Best et al. have provided a protocols to combine platelet RNA sequencing and swarm intelligence-enhanced classification algorithm development for disease diagnostics (78). ...

Hunting for the ultimate liquid cancer biopsy - Let the TEP dance begin

Cell Communication and Signaling