Thrombopoietin induces the generation of distinct Stat1, Stat3, Stat5a and Stat5b homo- and heterodimeric complexes with different kinetics in human platelets.
OBJECTIVE
Thrombopoietin (TPO) is the pivotal regulator of thrombocytopoiesis and megakaryocytopoiesis, and binding to its receptor c-Mpl leads to activation of at least two different signaling pathways: the Jak-Stat pathway and the Ras-MAPK pathway. Our aim was to elucidate which Stat-complexes are formed in TPO signal transduction in human blood platelets.


METHODS
We used electrophoretic mobility shift assays (EMSA) in order to analyze the formation of distinct Stat complexes on two distinct oligonucleotide probes. Furthermore, we used immunoprecipitation and Western blotting of protein lysates from TPO-stimulated platelets.


RESULTS
We found homodimers of Stat1alpha, Stat3, Stat5a, and Stat5b, as well as heterodimers of Stat1/Stat3 and Stat5a/Stat5b, but no Stat1/Stat5 or Stat3/Stat5 heterodimers are formed in platelets in response to TPO. Stat5 complexes bound to labeled DNA with a fast kinetic followed by Stat3 and Stat1. The adapter protein CrkL is present in DNA-bound Stat5 complexes and predominantly bound to Stat5b. The kinase ERK2 is also tyrosine phosphorylated after TPO-stimulation of platelets but this activation does not modulate the phosphorylation of the serine residues in the PXSP motif present in Stat1 and Stat3.


CONCLUSIONS
Our findings thus emphasize the differential regulation of Stat1, Stat3, Stat5a, and Stat5b in platelets and may be an appropriate model of c-Mpl signaling in mega-karyopoiesis.
Schulze
H
H
Department of Pediatric Hematology and Oncology, Medical School Hannover, Hannover, Germany.
Ballmaier
M
M
Welte
K
K
Germeshausen
M
M
D001792:Blood Platelets / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D019281:Dimerization; D006801:Humans; D008894:Milk Proteins; D015539:Platelet Activation / Q000187:drug effects*; D011485:Protein Binding; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects; D013926:Thrombopoietin / Q000494:pharmacology*; D015534:Trans-Activators / Q000737:chemistry / Q000502:physiology*; D025521:Tumor Suppressor Proteins
D004268:DNA-Binding Proteins; D008894:Milk Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; C494092:STAT5A protein, human; C494095:STAT5B protein, human; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D013926:Thrombopoietin