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, Adam N. Goldfarb

Research output: Contribution to journalArticle

24 Citations (Scopus)

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-ε (PKC-ε) 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 K562ΔRafER.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 ΔRafER 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.

Original languageEnglish (US)
Pages (from-to)1744-1751
Number of pages8
JournalBlood
Volume101
Issue number5
DOIs
StatePublished - Mar 1 2003
Externally publishedYes

Fingerprint

Guanine Nucleotide Exchange Factors
MAP Kinase Signaling System
Guanosine
Proto-Oncogene Proteins c-hck
Estradiol
Up-Regulation
Chemical activation
Proto-Oncogene Proteins c-raf
Extracellular Signal-Regulated MAP Kinases
Mutant Proteins
Stromal Cells
Coculture Techniques
Mitogen-Activated Protein Kinases
Protein Kinases
Protein Kinase C
Proteins
Refractory materials
Assays

ASJC Scopus subject areas

  • Hematology

Cite this

Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation. / Delehanty, Lorrie L.; Mogass, Michael; Gonias, Sara L.; Racke, Frederick K.; Johnstone, Brian; Goldfarb, Adam N.

In: Blood, Vol. 101, No. 5, 01.03.2003, p. 1744-1751.

Research output: Contribution to journalArticle

Delehanty, Lorrie L. ; Mogass, Michael ; Gonias, Sara L. ; Racke, Frederick K. ; Johnstone, Brian ; Goldfarb, Adam N. / Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation. In: Blood. 2003 ; Vol. 101, No. 5. pp. 1744-1751.
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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-ε (PKC-ε) 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 K562ΔRafER.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 ΔRafER 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.",
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