Anti-PEBP1 (aa 175-187) polyclonal antibody

Terminal erythroid differentiation is tightly coordinated with cell-cycle exit, which is regulated by cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain to be fully defined. We show here that p19 (INK4d,) a member of CDKI family, is abundantly expressed in erythroblasts and that p19 (INK4d) knockdown delayed erythroid differentiation, inhibited cell growth, and led to increased apoptosis and generation of abnormally nucleated late-stage erythroblasts. Unexpectedly, p19 (INK4d) knockdown did not affect cell cycle. Rather, it led to decreased expression of GATA1 protein. Importantly, the differentiation and nuclear defects were rescued by ectopic expression of GATA1. Because the GATA1 protein is protected by nuclear heat shock protein family (HSP) member HSP70, we examined the effects of p19 (INK4d) knockdown on HSP70 and found that p19 (INK4d) knockdown led to decreased expression of HSP70 and its nuclear localization. The reduced levels of HSP70 are the result of reduced extracellular signal-regulated kinase (ERK) activation. Further biochemical analysis revealed that p19 (INK4d) directly binds to Raf kinase inhibitor PEBP1 and that p19 (INK4d) knockdown increased the expression of PEBP1, which in turn led to reduced ERK activation. Thus we have identified an unexpected role for p19 INK4d via a novel PEBP1p- ERK-HSP70-GATA1 pathway. These findings are likely to have implications for improved understanding of disordered erythropoiesis.

Raf kinase inhibitory protein (RKIP/PEBP1), a member of the phosphatidylethanolamine binding protein family that possesses a conserved ligand-binding pocket, negatively regulates the mammalian mitogen-activated protein kinase (MAPK) signaling cascade. Mutation of a conserved site (P74L) within the pocket leads to a loss or switch in the function of yeast or plant RKIP homologues. However, the mechanism by which the pocket influences RKIP function is unknown. Here we show that the pocket integrates two regulatory signals, phosphorylation and ligand binding, to control RKIP inhibition of Raf-1. RKIP association with Raf-1 is prevented by RKIP phosphorylation at S153. The P74L mutation increases kinase interaction and RKIP phosphorylation, enhancing Raf-1/MAPK signaling. Conversely, ligand binding to the RKIP pocket inhibits kinase interaction and RKIP phosphorylation by a noncompetitive mechanism. Additionally, ligand binding blocks RKIP association with Raf-1. Nuclear magnetic resonance studies reveal that the pocket is highly dynamic, rationalizing its capacity to interact with distinct partners and be involved in allosteric regulation. Our results show that RKIP uses a flexible pocket to integrate ligand binding-and phosphorylation-dependent interactions and to modulate the MAPK signaling pathway. This mechanism is an example of an emerging theme involving the regulation of signaling proteins and their interaction with effectors at the level of protein dynamics.