Syk inhibits the activity of protein kinase a by phosphorylating tyrosine 330 of the catalytic subunit
Abstract
The Syk protein-tyrosine kinase can have multiple effects on cancer cells, acting in some as a tumor suppressor by inhibiting motility and in others as a tumor promoter by enhancing survival. Phosphoproteomic analyses identified protein kinase A (PKA) as a Syk-specific substrate. Syk catalyzes the phosphorylation of the catalytic subunit of PKA (PKAc) both in vitro and in cells on tyrosine 330 (Y330). Y330 lies within the adenosine binding motif in the carboxyl-terminal tail of PKAc within a cluster of acidic amino acids (DDYEEEE), which is a characteristic of Syk substrates. The phosphorylation of PKAc on Y330 by Syk strongly inhibits its catalytic activity. Molecular dynamics simulations suggest that this additional negative charge prevents the C-terminal tail from interacting with the substrate and the nucleotide binding site to stabilize the closed conformation of PKAc, thus preventing catalysis from occurring. Phosphoproteomic analyses and Western blotting studies indicate that Y330 can be phosphorylated in a Syk-dependent manner in MCF7 breast cancer cells and DT40 B cells. The phosphorylation of a downstream substrate of PKAc, CREB, is inhibited in cells expressing Syk, but can be rescued by a selective inhibitor of Syk. Modulation of CREB activity alters the expression of the CREB-regulated gene BCL2 and modulates cellular responses to genotoxic agents. Thus, PKA is a novel substrate of Syk and its phosphorylation on Y330 inhibits its participation in downstream signaling pathways. Immunofluorescence experiments show that in MCF7 and DT40 cells expressing Syk-EFGP-NLS, a form of fluorescently tagged Syk that has the nuclear localization sequence, PKKKRKV, placed at the C-terminus, the fusion protein is primarily nuclear, but the most abundant sites of tyrosine-phosphorylation are found in cytoplasmic vesicles identified as peroxisomes. Syk-EGFP-NLS also localizes partly to peroxisomes. Phosphoproteomic screening shows that ectopic expression of Syk-EGFP-NLS leads to the phosphorylation of multiple peroxisomal proteins in MCF7 cells. The NLS sequence, PKKKRKV, which is derived from SV40 large T antigen, directs Syk-EGFP-NLS into peroxisomes only when located at the C-terminus. Acox1, which is the oxidase that catalyzes the first step of β oxidation of very long chain fatty acids, is phosphorylated by Syk-EGFP-NLS and its enzymatic activity is decreased in cells expressing Syk-EGFP-NLS. This study shows that PKKKRKV doubles as a nuclear and peroxisomal targeting signal in eukaryotic cells. Thus, Syk-EGFP-NLS can be delivered into peroxisomes by this mechanism where it actively phosphorylates proteins located within these vesicles, which can lead to changes in their activities.
Degree
Ph.D.
Advisors
Geahlen, Purdue University.
Subject Area
Molecular biology|Pharmacology
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