Human Raf‐1 proteins associate with Rad24 and Cdc25 in cell‐cycle checkpoint pathway of fission yeast, Schizosaccharomyces pombe

Abstract

Raf-1 is a serine/threonine protein kinase that connects cell surface receptor signals to nuclear transcription factors. By screening Schizosaccharomyces pombe (S. pombe) cDNA library, we isolated Rad24, which is a 14-3-3 homolog that is important in the DNA damage checkpoint in S. pombe, as a Raf-1 interacting protein. The interaction found in yeast was confirmed by co-immunoprecipitation. Furthermore, Cdc25, which has been known to bind to Rad24, also associated with Raf-1 and was phosphorylated in vitro by catalytically active Raf-1. However, in the presence of Raf-1, an interaction between Rad24 and Cdc25 was inhibited in triple hybrid assay, indicating that Raf-1 inhibits the interaction between Rad24 and Cdc25. An in vitro competition assay showed that the binding of Cdc25 and of Rad24 to Raf-1 is mutually exclusive. Western blots of whole cell lysates probed with polyclonal antibodies specific for tyrosine-15-phosphorylated Cdc2 showed that overproduction of Rad24 led to the dephosphorylation of tyrosine residue on Cdc2, which is known to be activated through dephosphorylation by Cdc25 phosphatase. Unexpectedly, overexpression of catalytically inactive mutant protein of Raf-1, S624A, also caused tyrosine dephosphorylation of Cdc2. Thus, these data suggest that Raf-1 may interfere with the role of Rad24 by competing with Rad24 for binding to Cdc25 or a direct phosphorylation of Cdc25, bypassing the checkpoint pathway in DNA repair through Cdc25 activation.

Pharmacologic Inactivation of Kinase Suppressor of Ras1 Sensitizes Epidermal Growth Factor Receptor and Oncogenic Ras-Dependent Tumors to Ionizing Radiation Treatment

“…Only until recently experimental evidence has begun to illustrate a direct interaction of the Raf/ MEK/ERK signaling module (5,13,14,16,17,47,48), as well as EGFR signaling with DNA damage repair response (8)(9)(10)(11)(12). Although nuclear-localized EGFR direct interacts with DNA damage repair enzyme DNAdependent protein kinase catalytic subunit (DNA-PKcs), Raf-ERK signaling may regulate G 2 -M checkpoint and DNA damage repair through induction of critical genes participating in these processes, including GADD45 (14), XRCC1, and ERCC1 (5).…”

“…Although nuclear-localized EGFR direct interacts with DNA damage repair enzyme DNAdependent protein kinase catalytic subunit (DNA-PKcs), Raf-ERK signaling may regulate G 2 -M checkpoint and DNA damage repair through induction of critical genes participating in these processes, including GADD45 (14), XRCC1, and ERCC1 (5). Additionally, c-Raf-1 can associate with both 14-3-3 and Cdc25, connecting mitogenic signaling to cell-cycle checkpoint regulation (13). Moreover, BRAF mutation is linked to the proficiency of colorectal tumors in repairing mismatched bases in DNA (16,47).…”

“…In cancer cells, the Raf/MEK/ERK module is coupled to gain-of function (gf) Ras signaling in response to radiation (5,7). There is emerging molecular evidence linking this signaling cascade with checkpoint pathway (13)(14)(15), mismatch repair process (16), and DNA damage repair (5,17). However, upstream regulators that mediate the interaction between EGFR/Ras and Raf/MEK/ERK remain poorly characterized.…”

Overexpression of human Raf‐1 enhances radiosensitivity in fission yeast, Schizosaccharomyces pombe

“…10 Furthermore, Cdc25, which is known to bind to Rad24, also associated with Raf-1 and was phosphorylated in vitro by catalytically active Raf-1. Experiments were performed to determine the effect of overexpression of Raf-1 proteins on asynchronously growing S. pombe cells.…”

“…Recently, we found by twohybrid screening and co-immunoprecipitation experiments that Rad24, which is a 14-3-3 homologue that is important in the DNA damage checkpoint in Schizosaccharomyces pombe (S. pombe), 9 associated with Raf-1. 10 In S. pombe, Rad24 acts downstream of Chk1 in the DNA damage checkpoint and Rad24 null cells enter mitosis prematurely resulting in a small cell size at division. 9,11,12 Rad24 is thought to function as an attachable nuclear export signal that enhances the 13 The results presented here suggest that Raf-1 may interfere with the role of Rad24 by competing with Rad24 for binding to Cdc25 in DNA repair, bypassing the checkpoint pathway through Cdc25 activation.…”