Abstract
Protein kinases are implicated in multiple diseases such as cancer, diabetes, cardiovascular diseases, and central nervous system disorders. Identification of kinase substrates is critical to dissecting signaling pathways and to understanding disease pathologies. However, methods and techniques used to identify bona fide kinase substrates have remained elusive. Here we describe a proteomic strategy suitable for identifying kinase specificity and direct substrates in high throughput. This approach includes an in vitro kinase assay-based substrate screening and an endogenous kinase dependent phosphorylation profiling. In the in vitro kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the kinase of interest is added. Quantitative proteomics identifies the rephosphorylated proteins as direct substrates in vitro. In parallel, the in vivo quantitative phosphoproteomics is performed in which cells are treated with or without the kinase inhibitor. Together, proteins phosphorylated in vitro overlapping with the kinase-dependent phosphoproteome in vivo represents the physiological direct substrates in high confidence. The protein kinase assay-linked phosphoproteomics was applied to identify 25 candidate substrates of the protein-tyrosine kinase SYK, including a number of known substrates and many novel substrates in human B cells. These shed light on possible new roles for SYK in multiple important signaling pathways. The results demonstrate that this integrated proteomic approach can provide an efficient strategy to screen direct substrates for protein tyrosine kinases.
Highlights
Besides classical biochemical and genetic methods, mass spectrometry-based high throughput approaches have become increasingly attractive because they are capable of sequencing proteins and localizing phosphorylation sites at the same time
Analog-Sensitive Kinase Allele (ASKA) has recently been coupled with quantitative proteomics, termed Quantitative Identification of Kinase Substrates (QIKS) [12], to identify substrate proteins of Mek1
We address the shortcoming by introducing an alternative strategy termed Protein Kinase Assay-Linked Phosphoproteomics
Summary
Besides classical biochemical and genetic methods, mass spectrometry-based high throughput approaches have become increasingly attractive because they are capable of sequencing proteins and localizing phosphorylation sites at the same time. Taking advantage of recent advances of high speed and high-resolution mass spectrometry, these methods used purified, active kinases to phosphorylate cell extracts in vitro, followed by mass spectrometric analysis to identify phosphoproteins These approaches commonly face the major challenge of distinguishing phosphorylation events triggered by the kinase reaction from background signals introduced by endogenous kinase activities [20]. In addition to ASKA, radioisotope labeling using [␥-32P]ATP [10], using concentrated purified kinase [25], inactivating endogenous kinase activity by an additional heating step [11], and quantitative proteomics [26, 27] are alternative means aimed to address the same issues All of these methods, have been limited to the identification of in vitro kinase substrates. Coupled with in vivo phosphoproteomics, proKALIP has gained a high sensitivity and provided physiologically relevant substrates with high confidence
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