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Prepublished online as a Blood First Edition Paper on September 5, 2002; DOI 10.1182/blood-2002-04-1278.
Submitted April 30, 2002
Accepted August 23, 2002
Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation
Lorrie L Delehanty, Michael Mogass, Sara L Gonias, Frederick K Racke, Brian Johnstone, and Adam N Goldfarb*
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, VA, USA
Department of Pathology, Johns Hopkins Medical Institutions, Baltimore, MD, USA
Department of Orthopedics, Case Western Reserve University, Cleveland, OH, USA
* Corresponding author; email: ang3x{at}virginia.edu.
Co-culture with stromal cells tends to maintain normal hematopoietic progenitors and their leukemic counterparts in an undifferentiated, proliferative state. A dramatic example of this effect is seen with megakaryocytic differentiation, wherein stromal contact renders a variety of cell systems completely refractory to potent induction stimuli. This inhibitory effect of stroma on megakaryocytic differentiation appears to correlate with a blockade within hematopoietic cells of protein kinase C- (PKC-) upregulation and of 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 GTPase Rap1 was considered as a candidate inhibitory target. Activation assays confirmed that Rap1 underwent sustained activation as a result of megakaryocytic induction, as has been 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 upregulation but rather may target upstream guanine nucleotide exchange factor (GEF) complexes. Accordingly, co-immunoprecipitation demonstrated stromally induced alterations in a protein complex associated with c-Cbl, a scaffolding factor for Rap1-GEF complexes.
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