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Structural Identification of Major Molecular Determinants for Phosphotyrosine Recognition in Tyrosine Kinases Reveals
Nuo Cheng1, Luis R Millán-Barea2, Marc Creixell2
1Cancer Research UK Cambridge Institute, University of Cambridge, UK.
Abstract:
Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27Kip1. We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs - but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.
Insights
Protein kinases use phosphopriming to create twin phospho-tyrosine (pTyr) sites, crucial for cell cycle control. This mechanism, involving specific recognition sites, impacts cancer progression and leukemia development.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Protein tyrosine kinases (PTKs) regulate cellular signaling via tyrosine phosphorylation.
- PTKs can recognize phosphorylated tyrosine (pTyr) residues and catalyze adjacent phosphorylation, forming twin pTyr sites.
- This 'phosphopriming' mechanism is increasingly recognized as important in signaling pathways.
Purpose of the Study:
- To investigate the role of phosphopriming-driven twin pTyr signaling in cell cycle regulation using the protein p27Kip1.
- To identify and characterize the molecular determinants responsible for selective pTyr recognition adjacent to the phosphorylation site.
- To explore the implications of phosphopriming recognition in cancer and leukemia.
Main Methods:
- Structural biology (X-ray crystallography) to resolve protein-substrate interactions.
- Biochemical assays to assess kinase activity and substrate recognition.
- Site-directed mutagenesis to alter and test molecular determinants.
- In vivo studies using a mouse model of leukemia.
Main Results:
- Demonstrated the importance of phosphopriming in p27Kip1 phosphorylation and cell cycle progression.
- Identified and structurally characterized two key determinants for N- and C-terminal pTyr recognition.
- Showed conserved structural and biochemical features of this recognition across different kinases.
- Identified cancer-associated mutations that impair phosphopriming recognition.
- Observed paradoxical enhanced tumor development in a leukemia model with Bcr-Abl mutants lacking phosphopriming recognition.
Conclusions:
- Phosphopriming is a critical mechanism for regulating cell cycle proteins like p27Kip1.
- Specific molecular determinants govern the selective recognition of phosphoprimed substrates.
- Dysregulation of phosphopriming recognition is linked to cancer and can influence oncogenic signaling.
- Bcr-Abl utilizes both canonical and phosphopriming substrate recognition to modulate oncogenic programs.
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