Src inhibitors, PP2 and dasatinib, increase retinoic acid-induced association of Lyn and c-Raf (S259) and enhance MAPK-dependent differentiation of myeloid leukemia cells

Abstract

All-trans-retinoic-acid (ATRA)-induced differentiation of human myeloid leukemia cells is characterized by persistent MAPK signaling. Fragmentary data suggests Src family kinase (SFK) inhibitors enhance differentiation and thus have potential therapeutic value. The present study shows that SFK inhibitors PP2 and dasatinib enhance aspects of MAPK signaling and regulate a panel of differentiation markers including CD11b and p47phox. HL-60 and NB4 myeloid leukemia cells show accelerated ATRA-induced G1/0 arrest/differentiation with inhibitor co-treatment. We also identified components of a Lyn- and c-Raf-containing MAPK signaling complex augmented by the inhibitors. PP2 and dasatinib increased ATRA-induced expression of Lyn and c-Raf (total and c-RafpS259) and their interaction. The Lyn-associated serine/threonine kinase CK2 also complexed with c-Raf and c-RafpS259, and the KSR1 scaffold protein bound c-Raf, Lyn, and ERK. c-Raf/ERK association was increased by the inhibitors, which is significant since ERK may cause c-Raf C-terminal domain (CTD) phosphorylation in a putative feedback mechanism. Consistent with this, inhibitor treatment caused more CTD phosphorylation. Lyn knockdown decreased c-Raf CTD and S259 phosphorylation. This is the first evidence suggesting SFK inhibitors enhance ATRA-induced differentiation through a possible feedback loop involving KSR1-scaffolded c-Raf and ERK complexed with Lyn and CK2.

EGFR inhibitors exacerbate differentiation and cell cycle arrest induced by retinoic acid and vitamin D3in acute myeloid leukemia cells

“…40,41 Immunoblotting revealed that erlotinib and gefitinib inhibit the activating phosphorylation of p38 MAPK and SFKs, yet fail to suppress that of ERK and MAPK8 (best known as c-JUN N-terminal kinase 1, JNK1) (Fig. 6A).…”

Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons

“…To determine the effects of the SFK inhibitors on SFK signaling pathways, we examined the phosphorylation patterns of downstream effectors (Ceppi et al 2009; Congleton et al 2012). These data indicated that the SFK inhibitors do indeed affect SFK-mediated signaling pathways in motor neurons that are crucial for many cellular mechanisms (Fig.…”

GW5074 and PP2 kinase inhibitors implicate nontraditional c-Raf and Lyn function as drivers of retinoic acid-induced maturation

“…An RA-inducible interaction between the Src-family kinase (SFK) Lyn and pS259c-Raf was reported by Congleton et al (2012) [24]. Lyn and Fgr are the predominant SFKs in myeloid cells [31,32] and both are upregulated by RA treatment in HL-60 cells [11,24].…”

“…c-Raf propels RA-induced differentiation [12,19] but induced phosphorylation at the c-Raf activating sites S338 and Y340/Y341 cannot be detected in RA-treated HL-60 [20,21]. Instead, phosphorylation occurs at the S259 putative inhibitory site [22], the S621 putative stability site [23] and the S289/296/301 c-Raf sites [24,25]. The S289/296/301c-Raf sites are targets of ERK, but whether these sites are inhibitory [26] or activating [27] remains unclear (see Discussion).…”

“…Lyn and Fgr are the predominant SFKs in myeloid cells [31,32] and both are upregulated by RA treatment in HL-60 cells [11,24]. Lyn displays RA-inducible phosphorylation at both its activating SFK Y416 site, which on Lyn is Y396 [24,33] and its inhibitory Y507 site [34].…”