Mass Spectrometry-Based Discovery of &lt;i&gt;in vitro&lt;/i&gt; Kinome Substrates

et al. 2020

IJMS

Pancreatic cancer remains one of the most difficult malignancies to treat. Minimal improvements in patient outcomes and persistently abysmal patient survival rates underscore the great need for new treatment strategies. Currently, there is intense interest in therapeutic strategies that target tyrosine protein kinases. Here, we employed kinome arrays and bioinformatic pipelines capable of identifying differentially active protein tyrosine kinases in different patient-derived pancreatic ductal adenocarcinoma (PDAC) cell lines and wild-type pancreatic tissue to investigate the unique kinomic networks of PDAC samples and posit novel target kinases for pancreatic cancer therapy. Consistent with previously described reports, the resultant peptide-based kinome array profiles identified increased protein tyrosine kinase activity in pancreatic cancer for the following kinases: epidermal growth factor receptor (EGFR), fms related receptor tyrosine kinase 4/vascular endothelial growth factor receptor 3 (FLT4/VEGFR-3), insulin receptor (INSR), ephrin receptor A2 (EPHA2), platelet derived growth factor receptor alpha (PDGFRA), SRC proto-oncogene kinase (SRC), and tyrosine kinase non receptor 2 (TNK2). Furthermore, this study identified increased activity for protein tyrosine kinases with limited prior evidence of differential activity in pancreatic cancer. These protein tyrosine kinases include B lymphoid kinase (BLK), Fyn-related kinase (FRK), Lck/Yes-related novel kinase (LYN), FYN proto-oncogene kinase (FYN), lymphocyte cell-specific kinase (LCK), tec protein kinase (TEC), hemopoietic cell kinase (HCK), ABL proto-oncogene 2 kinase (ABL2), discoidin domain receptor 1 kinase (DDR1), and ephrin receptor A8 kinase (EPHA8). Together, these results support the utility of peptide array kinomic analyses in the generation of potential candidate kinases for future pancreatic cancer therapeutic development.

Section: Discussionmentioning

confidence: 99%

Kinome Array Profiling of Patient-Derived Pancreatic Ductal Adenocarcinoma Identifies Differentially Active Protein Tyrosine Kinases

Creeden

Alganem

et al. 2020

IJMS

“…Mass spectrometry (MS)-based phosphoproteomics has made it possible to identify thousands of phosphorylated sites in single experiments (1)(2)(3). However, despite the fact that enormous phosphosite information has been accumulated in public repositories (4-7), protein kinase-substrate relationships remain largely unknown both in vitro and in vivo (8).…”

Section: Introductionmentioning

confidence: 99%

“…Protein phosphorylation plays a key role in intracellular signal transduction and regulates various biological processes, including cell proliferation and differentiation. Mass spectrometry (MS)-based phosphoproteomics has made it possible to identify thousands of phosphorylated sites in single experiments ( 1 , 2 , 3 ). However, despite the fact that enormous phosphosite information has been accumulated in public repositories ( 4 , 5 , 6 , 7 ), protein kinase–substrate relationships remain largely unknown both in vitro and in vivo ( 8 ).…”

mentioning

confidence: 99%

Identification of Endogenous Kinase Substrates by Proximity Labeling Combined with Kinase Perturbation and Phosphorylation Motifs

Niinae

Molecular & Cellular Proteomics

Sugiyama

et al. 2021

Self Cite

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

“…Mass spectrometry (MS)-based phosphoproteomics has allowed us to identify thousands of phosphorylated sites in single experiments (1)(2)(3). However, despite the fact that enormous phosphosite information has been accumulated in public repositories (4)(5)(6)(7), protein kinase-substrate relationships remain largely unknown both in vitro and in vivo (8).…”

Section: Introductionmentioning

confidence: 99%

Proximity Labeling-assisted Identification of Endogenous Kinase Substrates

Niinae

Sugiyama

et al. 2020

Preprint

Self Cite

Mass spectrometry-based phosphoproteomics can identify more than 10,000 phosphorylated sites in a single experiment. But, despite the fact that enormous phosphosite information has been accumulated in public repositories, protein kinase-substrate relationships remain largely unknown. Here, we describe a method to identify endogenous substrates of kinases by means of proximity labeling. We used a proximity-dependent biotin identification approach, called BioID, in combination with kinase-perturbed phosphoproteomics profiling and phosphorylation sequence motifs derived from in vitro kinase assay to find molecules that interact with a target kinase, that show altered phosphorylation in response to kinase perturbation, and that are directly phosphorylated by the kinase in vitro; i.e., endogenous kinase substrates. Application of this methodology to casein kinase 2 (CK2) and protein kinase A (PKA) identified 33 and 52 putative substrates, respectively. We also show that known cancer-associated missense mutations near phosphosites of substrates affect phosphorylation by CK2 or PKA, and thus might alter downstream signaling in cancer cells bearing these mutations.This study extends our knowledge of kinase-substrate networks by proposing a new large-scale approach to identify endogenous substrates of kinases.