Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation

Abstract

Coculture with stromal cells tends to maintain normal hematopoietic progenitors and their leukemic counterparts in an undifferentiated, proliferative state. An example of this effect is seen with megakaryocytic differentiation, wherein stromal contact renders many cell types refractory to potent induction stimuli. This inhibitory effect of stroma on megakaryocytic differentiation correlates with a blockade within hematopoietic cells of protein kinase C-⑀ (PKC-⑀) up-regulation and of extracellular signal-regulated kinase/mitogen-activated protein (ERK/MAP) kinase activation, both of which have been implicated in promoting megakaryocytic differentiation. In this study K562⌬RafER.5 cells, expressing an estradiol-responsive mutant of the protein kinase Raf-1, were used to determine the relevance and stage of ERK/MAPK pathway blockade by stromal contact. Activation of ⌬RafER by estradiol overrode stromal blockade of megakaryocytic differentiation, implicating the proximal stage of the ERK/MAPK pathway as a relevant control point. Because stromal contact blocked delayed but not early ERK activation, the small guanosine triphosphatase (GTPase) Rap1 was considered as a candidate inhibitory target. Activation assays confirmed that Rap1 underwent sustained activation as a result of megakaryocytic induction, as previously described. As with ERK activation, stromal contact selectively blocked delayed but not early Rap1 activation, having no effect on Ras activation. Enforced expression of either wild-type Rap1 or the GTPase (GAP) resistant mutant Rap1 V12 failed to override stromal inhibition, suggesting that the inhibitory mechanism does not involve GAP up-regulation but rather may target upstream guanine nucleotide exchange factor (GEF) complexes. Accordingly, coimmunoprecipitation demonstrated stromally induced alterations in a protein complex associated with c-Cbl, a scaffolding factor for Rap1

Jun Blockade of Erythropoiesis: Role for Repression of GATA-1 by HERP2

“…on May 10, 2018 by guest http://mcb.asm.org/ protein in response to tetradecanoyl phorbol acetate and that the megakaryocytic differentiation of K562 cells in response to tetradecanoyl phorbol acetate is only partial (11). Nevertheless, it is possible that different thresholds for HERP2 inhibition of GATA function exist in erythroid versus megakaryocytic differentiation.…”

“…Immunoblot analysis of K562 wholecell lysates was conducted as previously described (11,14). GATA-1 was detected with rat monoclonal anti-GATA-1 (N6; Santa Cruz Biotechnology, Santa Cruz, Calif.).…”

“…Notably, tetradecanoyl phorbol acetate treatment also causes downregulation of the erythroid marker GPA in K562 cells (11). The question arises how K562 cells may undergo megakaryocytic differentiation in response to tetradecanoyl phorbol acetate when the upregulation of HERP2 is expected to block GATA factor function.…”

Dok-4 regulates GDNF-dependent neurite outgrowth through downstream activation of Rap1 and mitogen-activated protein kinase

“…In PC12 cells, an established model of NGFinduced neuronal differentiation, Rap1 is activated by NGF, which then increases the duration of ERK signaling by acting through the related Raf isoform B-Raf, thereby potentiating neuronal differentiation (Vossler et al, 1997;York et al, 1998;Annerén et al, 2000;Grewal et al, 2000b). In megakaryocytes, activation of Rap1, B-Raf, and ERK by thrombopoietin induces megakaryocytic differentiation (Delehanty et al, 2003).…”

Unraveling the mysteries of B-Raf: the clot thickens!

“…2 B-Raf, unlike Raf-1, can be activated by the small G protein Rap1, 3 and Rap1/BRaf signaling to ERKs has been implicated in this sustained activation of ERKs and differentiation by TPO. 2,4 These studies establish that B-Raf and Raf-1 appear to have lineage-specific functions. Recent studies demonstrate that Raf-1 is required for erythropoiesis, 5 but not megakaryopoiesis.…”