scholarly journals Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites

2018 ◽  
Author(s):  
Aaron J. Cantor ◽  
Neel H. Shah ◽  
John Kuriyan
Keyword(s):  
Tyrosine Kinases ◽  
Mutational Analysis ◽  
Signal Transmission ◽  
Surface Display ◽  
Growth Factor Receptor ◽  
Adapter Proteins ◽  
Hydrophobic Residues ◽  
Tyrosine Residues ◽  
Trade Offs ◽  
Ptb Domains

AbstractUpon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src Homology 2 (SH2) and Phosphotyrosine Binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as phosphosites) must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. In order to understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface-display of peptides, coupled with deep sequencing, to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the −1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins, but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation while maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.

scholarly journals Deep mutational analysis reveals functional trade-offs in the sequences of EGFR autophosphorylation sites

2018 ◽  
Vol 115 (31) ◽  
pp. E7303-E7312 ◽  
Author(s):  
Aaron J. Cantor ◽  
Neel H. Shah ◽  
John Kuriyan
Keyword(s):  
Tyrosine Kinases ◽  
Mutational Analysis ◽  
Signal Transmission ◽  
Surface Display ◽  
Growth Factor Receptor ◽  
Adapter Proteins ◽  
Hydrophobic Residues ◽  
Tyrosine Residues ◽  
Trade Offs ◽  
Ptb Domains

Upon activation, the epidermal growth factor receptor (EGFR) phosphorylates tyrosine residues in its cytoplasmic tail, which triggers the binding of Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains and initiates downstream signaling. The sequences flanking the tyrosine residues (referred to as “phosphosites”) must be compatible with phosphorylation by the EGFR kinase domain and the recruitment of adapter proteins, while minimizing phosphorylation that would reduce the fidelity of signal transmission. To understand how phosphosite sequences encode these functions within a small set of residues, we carried out high-throughput mutational analysis of three phosphosite sequences in the EGFR tail. We used bacterial surface display of peptides coupled with deep sequencing to monitor phosphorylation efficiency and the binding of the SH2 and PTB domains of the adapter proteins Grb2 and Shc1, respectively. We found that the sequences of phosphosites in the EGFR tail are restricted to a subset of the range of sequences that can be phosphorylated efficiently by EGFR. Although efficient phosphorylation by EGFR can occur with either acidic or large hydrophobic residues at the −1 position with respect to the tyrosine, hydrophobic residues are generally excluded from this position in tail sequences. The mutational data suggest that this restriction results in weaker binding to adapter proteins but also disfavors phosphorylation by the cytoplasmic tyrosine kinases c-Src and c-Abl. Our results show how EGFR-family phosphosites achieve a trade-off between minimizing off-pathway phosphorylation and maintaining the ability to recruit the diverse complement of effectors required for downstream pathway activation.

scholarly journals Mutational analysis of Lck in CD45-negative T cells: dominant role of tyrosine 394 phosphorylation in kinase activity.

1996 ◽  
Vol 16 (9) ◽  
pp. 4996-5003 ◽  
Author(s):  
U D'Oro ◽  
K Sakaguchi ◽  
E Appella ◽  
J D Ashwell
Keyword(s):  
T Cells ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Mutational Analysis ◽  
Dominant Role ◽  
Tyrosine Phosphatase ◽  
Double Mutation ◽  
Tyrosine Residues ◽  
Lck Kinase

The CD45 tyrosine phosphatase has been reported to activate the src family tyrosine kinases Lck and Fyn by dephosphorylating regulatory COOH-terminal tyrosine residues 505 and 528, respectively. However, recent studies with CD45- T-cell lines have found that despite the fact that Lck and Fyn were constitutively hyperphosphorylated, the tyrosine kinase activity of both enzymes was actually increased. In the present study, phosphoamino acid analysis revealed that the increased phosphorylation of Lck in CD45- YAC-1 T cells was restricted to tyrosine residues. To understand the relationship between tyrosine phosphorylation and Lck kinase activity, CD45- YAC-1 cells were transfected with forms of Lck in which tyrosines whose phosphorylation is thought to regulate enzyme activity (Tyr-192, Tyr-394, Tyr-505, or both Tyr-394 and Tyr-505) were replaced with phenylalanine. While the Y-to-F mutation at position 192 (192-Y-->F) had little effect, the 505-Y-->F mutation increased enzymatic activity. In contrast, the 394-Y-->F mutation decreased the kinase activity to very low levels, an effect that the double mutation, 394-Y-->F and 505Y-->F, could not reverse. Phosphopeptide analysis of tryptic digests of Lck from CD45- YAC-1 cells revealed that it is hyperphosphorylated on two tyrosine residues, Tyr-505 and, to a lesser extent, Tyr-394. The purified and enzymatically active intracellular portion of CD45 dephosphorylated Lck Tyr-394 in vitro. These results demonstrate that in addition to Tyr-505, CD45 can dephosphorylate Tyr-394, and that in the absence of CD45 the hyperphosphorylation of Tyr-394 can cause an increase in the kinase activity of Lck despite the inhibitory hyperphosphorylation of Tyr-505. Therefore, Lck kinase activity is determined by the balance of activating and inhibitory tyrosine phosphorylations that are, in turn, regulated by CD45.

scholarly journals Aging Regulated Through a Stability Model of Insulin/Insulin Growth Factor Receptor Function

2021 ◽  
Vol 12 ◽  
Author(s):  
Marc Tatar
Keyword(s):  
Growth Factor ◽  
Insulin Receptor ◽  
Tyrosine Kinases ◽  
Phosphorylation Site ◽  
Growth Factor Receptor ◽  
High Threshold ◽  
Insulin Growth Factor ◽  
Trade Offs ◽  
The Stability ◽  
New Research

Mutations of the insulin-like receptor in Drosophila extend lifespan. New research suggests this receptor operates in two modes. The first extends lifespan while slowing reproduction and reducing growth. The second strongly extends lifespan without impairing growth or reproduction; it confers longevity assurance. The mutation that confers longevity assurance resides in the kinase insert domain, which contains a potential SH2 binding site for substrate proteins. We apply a recent model for the function of receptor tyrosine kinases to propose how insulin receptor structure can modulate aging. This concept hypothesizes that strong insulin-like ligands promote phosphorylation of high threshold substrate binding sites to robustly induce reproduction, which impairs survival as a consequence of trade-offs. Lower levels of receptor stimulation provide less kinase dimer stability, which reduces reproduction and extends lifespan by avoiding reproductive costs. Environmental conditions that favor diapause alter the expression of insulin ligands to further repress the stability of the interacting kinase domains, block phosphorylation of low threshold substrates and thus induce a unique molecular program that confers longevity assurance. Mutations of the insulin receptor that block low-phosphorylation site interactions, such as within the kinase insert domain, can extend lifespan while maintaining overall dimer stability. These flies are long-lived while maintaining reproduction and growth. The kinase insert domain of Drosophila provides a novel avenue from which to seek signaling of the insulin/insulin-like growth factor system of humans that modulate aging without impacting reproduction and growth, or incurring insulin resistance pathology.

scholarly journals The intramolecular allostery of GRB2 governing its interaction with SOS1 is modulated by phosphotyrosine ligands

2021 ◽  
Author(s):  
Neda Sadat Kazemein Jasemi ◽  
Christian Herrmann ◽  
Eva Magdalena Estirado ◽  
Lothar Gremer ◽  
Dieter Willbold ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Important Advance ◽  
Tyrosine Residues ◽  
Rich Domain ◽  
Allosteric Mechanism ◽  
Domain Interactions ◽  
Growth Factor Receptor Bound

Growth factor receptor-bound protein 2 (GRB2) is a trivalent adaptor protein and a key element in signal transduction. It interacts via its flanking nSH3 and cSH3 domains with the proline-rich domain (PRD) of the RAS activator SOS1 and via its central SH2 domain with phosphorylated tyrosine residues of receptor tyrosine kinases (RTKs; e.g., HER2). The elucidation of structural organization and mechanistic insights into GRB2 interactions, however, remain challenging due to their inherent flexibility. This study represents an important advance in our mechanistic understanding of how GRB2 links RTKs to SOS1. Accordingly, it can be proposed that (1) HER2 pYP-bound SH2 potentiates GRB2 SH3 domain interactions with SOS1 (an allosteric mechanism); (2) the SH2 domain blocks cSH3,enabling nSH3 to bind SOS1 first before cSH3 follows (an avidity-based mechanism); and (3) the allosteric behavior of cSH3 to other domains appears to be unidirectional, although there is an allosteric effect between the SH2 and SH3 domains.

Platelet-derived growth factor receptor alpha (PDGFRA) and C-Kit immunohistochemistry (IHC) in gastrointestinal stromal tumours (GISTs)

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 15156-15156
Author(s):  
D. Skarlos ◽  
E. Kyrodimou ◽  
N. Papakonstantinou ◽  
G. Klouvas ◽  
C. Christodoulou ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Mutational Analysis ◽  
Growth Factor Receptor ◽  
Gastrointestinal Stromal Tumours ◽  
Exact Role ◽  
Male Predominance ◽  
Activating Mutations ◽  
Study Materials ◽  
Factor Receptor Alpha

15156 Background: The vast majority of GISTs show activating mutations of the genes coding for c-kit (CD 117) and PDGFRA, two receptor tyrosine kinases (RTKs). The exact role of PDGFRA as this expressed immunohistochemically (IHC), has not been fully elucidated in the diagnosis and treatment of GISTs. The aim of this study was (1) to try to correlate the pathological features of patients (pts) with GISTs, with c-kit and PDGFRA as these expressed by IHC and 2) to correlate the c-kit and PDGFRA with the clinical outcome of the pts. We report here the first part of the study. Materials and Methods: Twenty one (21) pts with GISTs were studied 14 located in the stomach, 3 in small bowel, 3 in colon and 1 in the mesenterium. Criteria for positivity of c-kit were strong membranus, cytoplasmic and paranuclear staining and for PDGFRA dot-like or Golgi pattern of staining. Median age was 61 years while there was a male predominance. Results: C-kit was detected in 95% of cases while PDGFRA in 90.5%. All the GISTs that were positive for PDGFRA were also positive for c-kit. Conclusions: Preliminary results of the first part of the study show that an accurate diagnosis of GISTs can be achieved based on the conventional histological and IHC criteria without the need for mutational analysis. No significant financial relationships to disclose.

The Biology of Epidermal Growth Factor Receptor and its Value as a Prognostic/predictive Factor

2007 ◽  
Vol 22 (1_suppl4) ◽  
pp. 3-9 ◽  
Author(s):  
A.P. Dei Tos
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Small Molecules ◽  
Tyrosine Kinases ◽  
Mutational Analysis ◽  
Growth Factor Receptor ◽  
Poor Clinical Outcome ◽  
Egfr Expression ◽  
Epidermal Growth

Receptor tyrosine kinases play a major role in human carcinogenesis. Overexpression of the epidermal growth factor receptor (EGFR) has been associated with poor clinical outcome in several types of cancer. In principle, as with HER2, the EGFR status of a tumor should predict the likelihood of response to EGFR-targeted therapy. However, clinical data have failed to demonstrate a relationship between EGFR expression and response to the EGFR-targeted compounds cetuximab, gefitinib and erlotinib. Recently, patients reported to be EGFR negative have been shown to respond to cetuximab. Possible explanations include methodological failures or most likely heterogeneity and complexity of the mechanisms of EGFR-mediated molecular carcinogenesis. Immunohistochemistry is the most widely used method for measuring EGFR expression; however, its value is limited by lack of methodological standardization. Other approaches to measuring EGFR such as amplification assays are currently being introduced but need further testing before they can enter clinical practice. Mutational analysis seems also fruitful in predicting response to anti-EGFR small molecules. Further work is needed to identify how EGFR contributes to the carcinogenic and metastatic processes.

Targeting Small Molecule Tyrosine Kinases by Polyphenols: New Move Towards Anti-tumor Drug Discovery

2020 ◽  
Vol 17 (5) ◽  
pp. 585-615 ◽  
Author(s):  
Nikhil S. Sakle ◽  
Shweta A. More ◽  
Sachin A. Dhawale ◽  
Santosh N. Mokale
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinase ◽  
Cancer Cells ◽  
Small Molecule ◽  
Anaplastic Lymphoma Kinase ◽  
Tyrosine Kinases ◽  
Growth Factor Receptor ◽  
Myelogenous Leukemia ◽  
Cell Functions ◽  
Anaplastic Lymphoma

Background: Cancer is a complex disease involving genetic and epigenetic alteration that allows cells to escape normal homeostasis. Kinases play a crucial role in signaling pathways that regulate cell functions. Deregulation of kinases leads to a variety of pathological changes, activating cancer cell proliferation and metastases. The molecular mechanism of cancer is complex and the dysregulation of tyrosine kinases like Anaplastic Lymphoma Kinase (ALK), Bcr-Abl (Fusion gene found in patient with Chronic Myelogenous Leukemia (CML), JAK (Janus Activated Kinase), Src Family Kinases (SFKs), ALK (Anaplastic lymphoma Kinase), c-MET (Mesenchymal- Epithelial Transition), EGFR (Epidermal Growth Factor receptor), PDGFR (Platelet-Derived Growth Factor Receptor), RET (Rearranged during Transfection) and VEGFR (Vascular Endothelial Growth Factor Receptor) plays major role in the process of carcinogenesis. Recently, kinase inhibitors have overcome many problems of traditional cancer chemotherapy as they effectively separate out normal, non-cancer cells as well as rapidly multiplying cancer cells. Methods: Electronic databases were searched to explore the small molecule tyrosine kinases by polyphenols with the help of docking study (Glide-7.6 program interfaced with Maestro-v11.3 of Schrödinger 2017) to show the binding energies of polyphenols inhibitor with different tyrosine kinases in order to differentiate between the targets. Results: From the literature survey, it was observed that the number of polyphenols derived from natural sources alters the expression and signaling cascade of tyrosine kinase in various tumor models. Therefore, the development of polyphenols as a tyrosine kinase inhibitor against targeted proteins is regarded as an upcoming trend for chemoprevention. Conclusion: In this review, we have discussed the role of polyphenols as chemoreceptive which will help in future for the development and discovery of novel semisynthetic anticancer agents coupled with polyphenols.

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