title: 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-epsilon (PKC-epsilon) 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 K562DeltaRafER.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 DeltaRafER 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-GEF complexes.
authors.affiliation: Department of Pathology, University of Virginia Health Sciences Center, Charlottesville, MD 22908, USA.
authors.forename: Lorrie L
authors.initials: LL
authors.lastname: Delehanty
authors.forename: Michael
authors.initials: M
authors.lastname: Mogass
authors.forename: Sara L
authors.initials: SL
authors.lastname: Gonias
authors.forename: Frederick K
authors.initials: FK
authors.lastname: Racke
authors.forename: Brian
authors.initials: B
authors.lastname: Johnstone
authors.forename: Adam N
authors.initials: AN
authors.lastname: Goldfarb
mesh_terms: D001854:Bone Marrow Cells / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D018920:Coculture Techniques; D003599:Cytoskeleton / Q000648:ultrastructure; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D046911:Macromolecular Substances; D008533:Megakaryocytes / Q000166:cytology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009363:Neoplasm Proteins / Q000502:physiology; D011518:Proto-Oncogene Proteins / Q000502:physiology; D050721:Proto-Oncogene Proteins c-cbl; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology; D015398:Signal Transduction; D017154:Stromal Cells / Q000502:physiology; D044767:Ubiquitin-Protein Ligases; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D011518:Proto-Oncogene Proteins; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D020827:rap1 GTP-Binding Proteins; C067419:CBL protein, human