Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

Abstract

Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G(1) phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

Pyk2 cytonuclear localization: mechanisms and regulation by serine dephosphorylation

“…Pyk2 could directly regulate tyrosine phosphorylated resident proteins of the nucleus such as histone variant H2AX [51] or H3/H4 [52]. Pyk2 associates with Src-family kinases, which localize to the nucleus in specific conditions, regulating euchromatin decondensation [53] and protection against oxidative stress through phosphorylation of Nrf2 by Fyn [54,55]. Nuclear Pyk2 may also play a role of a scaffolding protein for p53 [35].…”

Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint

“…For this purpose, the SFK member Lyn was tagged with a nuclear localization signal (NLS-Lyn) and transiently expressed in thymidine-treated cells. Transient expression of NLS-Lyn promotes protein tyrosine phosphorylation in the nucleus (43,55). Chk1-Ser 317 phosphorylation was induced by thymidine treatment and detected in flow cytometry (Fig.…”

“…FH-NLS-Lyn retains the inhibitory tyrosine phosphorylation site at the C-terminal tail. The constitutively active mutants Lyn⌬C-HA (deleted of residues 507-512) and NLS-Lyn⌬C-HA were described previously (36,43). To generate HeLa S3 cells with an inducible v-Src allele (HeLa S3-TR/v-Src), HeLa S3 cells were transfected with the tetracycline repressor (TR) and selected with hygromycin (HeLa S3-TR).…”

“…Binucleation Assay-HeLa S3 cells were synchronized at the G 1 /S boundary and exposed to 110 nM Adriamycin for 1 h. Then cells were released from synchronization and cultured in the presence of 0.2 g/ml cytochalasin D. After 23 h of recovery, cells were fixed with 4% paraformaldehyde and stained with propidium iodide as described previously (42,43). Binucleated cells were counted under a fluorescence microscope (Zeiss Axiovert S100).…”

“…For detection of nuclear localization of Lyn⌬C, the transfected cells were fixed with 4% paraformaldehyde. Subsequently, the cells were permeabilized with 0.5% Triton X-100 for 5 min and stained as described previously (42,43). DNA was stained with propidium iodide.…”