Identification of direct tyrosine kinase substrates based on protein kinase assay-linked phosphoproteomics

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.

Fig. 1.

Methodology of proKALIP to identify kinase substrates. proKALIP combines in vitro kinase reactions and in vivo phosphoproteomics. In the in vitro kinase reaction, substrate proteins are isolated from cell lysates through affinity purification, dephosphorylated by alkaline phosphatase, and rephosphorylated by kinase of interest. After an in vitro kinase reaction, phosphopeptides are further enriched and analyzed by mass spectrometry for sequencing and site identification. Theoretical substrate has a higher intensity in kinase+ sample compared with the control in SILAC experiments. In in vivo phosphoproteomics, kinase dependent phosphorylation events are identified by comparing two phosphoproteomes with kinase perturbations. Genuine substrates are the phosphopeptides present within both data sets from in vitro kinase reaction and in vivo phosphoproteomics.

Fig. 2.

Evaluation of FSBA inhibition under in vitro kinase reaction condition. A, Kinase assay with no exogenous kinase and FSBA was added in the light fraction. B, Kinase assay with the exogenous SYK and FSBA was added in the light fraction.

Fig. 3.

Ratio distributions of phosphopeptide intensity before and after kinase reaction using reciprocal SILAC quantifications. A, SYK was added in the heavy fraction, whereas light fraction was the control. B, SYK was added in the light fraction, whereas heavy fraction was the control. C, Dot-plot depicting the overlap of the reciprocal data sets of tyrosine phosphorylation sites identified using in vitro kinase reaction.

Fig. 4.

A, Consensus sequence analysis of in vitro kinase phosphorylation for SYK specificity. Upper panel, frequency plot of all amino acids flanking the phosphotyrosine site. Lower panel, significantly enriched motif from SYK substrates using Motif-X; B, Venn diagram illustrating the overlap of tyrosine phosphorylation sites identified by in vitro kinase reaction and in vivo phosphoproteomics in DG75 cells. C, Categories of biological processes for identified SYK substrates.

Fig. 5.

Western blotting for confirmation of SYK substrates through in vitro kinase assay and in vivo phosphorylation changes in response to BCR stimulation in DG-75 cells. For in vitro kinase assay, individual proteins were immunoprecipitated from cell lysates. The immune complex was then incubated with in a kinase reaction containing purified SYK kinase. For BCR stimulation, DG75 B cells were treated without (IgM-) or with (IgM+) anti-IgM antibodies. Individual proteins were directly isolated from cell lysates. The reaction mixtures were separated by SDS-PAGE and blotted with antigen antibodies and anti-phosphotyrosine antibody (4G10).