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Proteomic Analysis of Src Family Kinase Phosphorylation States in Cancer Cells Suggests Deregulation of the Unique Domain.

ABSTRACT: The Src family kinases (SFK) are homologs of retroviral oncogenes, earning them the label of proto-oncogenes. Their functions are influenced by positive and negative regulatory tyrosine phosphorylation events and inhibitory and activating intramolecular and extramolecular interactions. This regulation is disrupted in their viral oncogene counterparts. However, in contrast to most other proto-oncogenes, the genetic alteration of these genes does not seem to occur in human tumors and how and whether their functions are altered in human cancers remain to be determined. To look for proteomic-level alterations, we took a more granular look at the activation states of SFKs based on their two known regulatory tyrosine phosphorylations, but found no significant differences in their activity states when comparing immortalized epithelial cells with cancer cells. SFKs are known to have other less well-studied phosphorylations, particularly within their unstructured N-terminal unique domains (UD), although their role in cancers has not been explored. In comparing panels of epithelial cells with cancer cells, we found a decrease in S17 phosphorylation in the UD of Src in cancer cells. Dephosphorylated S17 favors the dimerization of Src that is mediated through the UD and suggests increased Src dimerization in cancers. These data highlight the important role of the UD of Src and suggest that a deeper understanding of proteomic-level alterations of the unstructured UD of SFKs may provide considerable insights into how SFKs are deregulated in cancers. IMPLICATIONS: This work highlights the role of the N-terminal UD of Src kinases in regulating their signaling functions and possibly in their deregulation in human cancers.

SUBMITTER: Ruiz-Saenz A

PROVIDER: S-EPMC8178206 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Src family kinases are often overexpressed and constitutively active in cancer cells, where they represent promising targets for therapeutic intervention. In myeloid hematopoietic cells, Hck, Lyn, and Fgr are associated with both chronic and acute forms of myeloid leukemia. All three of these kinases are expressed in acute myeloid leukemia (AML), where high-level expression correlates with poor survival. The pyrrolopyrimidine Src-family kinase inhibitor A-419259 (also known as RK-20449) has been shown to significantly inhibit patient-derived AML bone marrow cell growth in immunocompromised mice. Recent work has identified an N-phenylbenzamide kinase inhibitor, known as TL02-59, which potently inhibited Fgr kinase activity in vitro and reversed orthotopic engraftment of AML cells in the bone marrow and spleen of immunocompromised mice following oral administration. The inhibitory mechanism of this compound with Fgr or other Src-family members has not been reported. Both wild-type Fgr and a tail mutant (i.e., replacement of Tyr527 with Phe) were shown to produce equal numbers of transformed colonies when expressed in rodent fibroblasts, whereas wild-type Hck transforming activity was completely repressed. In both Hck and Fgr, however, the tail tyrosine residue was shown to be phosphorylated, and hydrogen deuterium exchange mass spectrometry (HDX MS) of near-full-length Fgr was consistent with the closed conformation in solution, with the SH3 domain bound to the SH2-kinase linker and the tail bound to SH2. Here we present the first X-ray crystal structures of near-full-length Fgr bound to the ATP site inhibitors A-419259 and TL02-59. Structural changes induced by the inhibitors in the crystalline state were validated in solution using HDX MS, with A-419259 stabilizing the closed conformation and TL02-59 enhancing exposure of the SH3 domain to solvent. These new structures help to explain the constitutive activity of wild-type Fgr in cells as well as its unique sensitivity to TL02-59, which is currently in development for AML and other maladies.

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Proteomic Analysis of Src Family Kinase Phosphorylation States in Cancer Cells Suggests Deregulation of the Unique Domain.

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