Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

Jin, Lily L.; Wybenga‐Groot, Leanne E.; Tong, Jiefei; Taylor, Paul; Minden, Mark D.; Trudel, Suzanne; McGlade, C. Jane; Moran, Michael F.

doi:10.1074/mcp.m114.044404

Cited by 34 publications

(33 citation statements)

References 50 publications

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“…Although the classical in-solution digestion protocol with urea lysis buffer has provided satisfactory phosphoproteomic results in previous AML studies [11,12,13], our comparison study showed that it did not perform as well as the in-solution digestion protocol with guanidinium hydrochloride lysis buffer and FASP. A previous comparison of different lysis buffers used in the in-solution digestion method also showed a poor protocol performance when using the urea lysis buffer [34].…”

Section: Resultsmentioning

confidence: 58%

“…Moreover, SH2 domains of Src family kinases are themselves tyrosine phosphorylated in some blood cancers including AML [13]. To study the binding properties of those tyrosine-phosphorylated SH2 domains to phosphotyrosine-containing polypeptides and proteins, peripheral blood mononuclear cells from 12 AML patients were collected and lysed with a buffer containing 9 M urea and sodium vanadate among several phosphate-containing molecules that served as phosphatase substrates.…”

Section: Sample Preparation Workflows In Global Ms Proteome Studiementioning

confidence: 99%

“…To study the binding properties of those tyrosine-phosphorylated SH2 domains to phosphotyrosine-containing polypeptides and proteins, peripheral blood mononuclear cells from 12 AML patients were collected and lysed with a buffer containing 9 M urea and sodium vanadate among several phosphate-containing molecules that served as phosphatase substrates. The trypsin digested samples were enriched for phosphotyrosine peptides using PhosphoScan reagents (Cell Signaling Technology, Danvers, MA, USA) and analyzed with a Q Exactive or Elite (Thermo Fisher Scientific) mass spectrometers [13]. Sixty phosphotyrosine peptides were identified with an adapted phospho-enrichment step using 2 mg of cell lysate [21].…”

Section: Sample Preparation Workflows In Global Ms Proteome Studiementioning

confidence: 99%

“…However, proteomic and phosphoproteomic workflows are not universal and they might work differently depending on the characteristics of the sample and the experience of the researcher. Published global MS proteome and phosphoproteome analysis of AML patient samples have used the standard one-dimensional electrophoresis (1DE), 2DE and the in-solution digestion method with urea in the lysis buffer to digest AML proteins prior to MS analysis or phosphorylation enrichment [10,11,12,13,14]. We have recently tested several methods of sample preparation for the MS analysis of the AML blasts that included in-solution digestion methods with urea and guanidinium hydrochloride as denaturant reagents in the lysis buffer and filter-aided sample preparation (FASP) [15,16].…”

Section: Introductionmentioning

confidence: 99%

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Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

et al. 2016

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Global mass spectrometry (MS)-based proteomic and phosphoproteomic studies of acute myeloid leukemia (AML) biomarkers represent a powerful strategy to identify and confirm proteins and their phosphorylated modifications that could be applied in diagnosis and prognosis, as a support for individual treatment regimens and selection of patients for bone marrow transplant. MS-based studies require optimal and reproducible workflows that allow a satisfactory coverage of the proteome and its modifications. Preparation of samples for global MS analysis is a crucial step and it usually requires method testing, tuning and optimization. Different proteomic workflows that have been used to prepare AML patient samples for global MS analysis usually include a standard protein in-solution digestion procedure with a urea-based lysis buffer. The enrichment of phosphopeptides from AML patient samples has previously been carried out either with immobilized metal affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC). We have recently tested several methods of sample preparation for MS analysis of the AML proteome and phosphoproteome and introduced filter-aided sample preparation (FASP) as a superior methodology for the sensitive and reproducible generation of peptides from patient samples. FASP-prepared peptides can be further fractionated or IMAC-enriched for proteome or phosphoproteome analyses. Herein, we will review both in-solution and FASP-based sample preparation workflows and encourage the use of the latter for the highest protein and phosphorylation coverage and reproducibility.

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Section: Resultsmentioning

confidence: 58%

Section: Sample Preparation Workflows In Global Ms Proteome Studiementioning

confidence: 99%

Section: Sample Preparation Workflows In Global Ms Proteome Studiementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

et al. 2016

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“…Previous investigations revealed that mutation or phosphorylation of Y192 has the potential to affect the affinity or specificity of the SH2 domain (Couture et al, 1996; Granum et al, 2014; Jin et al, 2015; Stover et al, 1996). One potential mode of SH2-dependent regulation would be the accessibility of the phosphorylated C-terminal tail (Model 1, Figure S4).…”

Section: Resultsmentioning

confidence: 99%

A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45

et al. 2017

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SUMMARY The Src Family kinase Lck sets a critical threshold for T cell activation because it phosphorylates the TCR complex and the Zap70 kinase. How a T cell controls the abundance of active Lck molecules remains poorly understood. We have identified an unappreciated role for a phosphosite, Y192, within the Lck SH2 domain which profoundly affects the amount of active Lck in cells. Notably, mutation of Y192 blocks critical TCR proximal signaling events and impairs thymocyte development in retrogenic mice. We determined that these defects are caused by hyperphosphorylation of the inhibitory C-terminal tail of Lck. Our findings reveal that modification of Y192 inhibits the ability of CD45 to associate with Lck in cells and dephosphorylate the C-terminal tail of Lck which prevents its adoption of an active open conformation. These results suggest a negative feedback loop which responds to signaling events that tune active Lck amounts and TCR sensitivity.

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Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain‐binding capacity

et al. 2016

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Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly-regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly-phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the site C-terminal to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain.

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Tyrosine Phosphorylation of the Lyn Src Homology 2 (SH2) Domain Modulates Its Binding Affinity and Specificity*

Cited by 34 publications

References 50 publications

Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

A Phosphosite within the SH2 Domain of Lck Regulates Its Activation by CD45

Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain‐binding capacity

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