Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1511-1512
CORRESPONDENCE
 |
Letter |
To the editor:
MAP kinase localizes to the platelet-yielding demarcation membrane
system in megakaryocytes
Megakaryopoiesis and increased platelet levels are
greatly promoted by the c-Mpl ligand, thrombopoietin (TPO), which binds to the c-Mpl receptor.1 Among the signal transduction
pathways emanating from the c-Mpl receptor is the Ras/MEK/MAP kinase
pathway.2 MAP kinases (also known as ERK) were originally
identified as the microtubule-associated
protein kinases by virtue of their ability to
phosphorylate microtubule-associated proteins.3-5 A recent
study by Lecine et al indicated that 
-tubulin is an essential
component for platelet fragmentation.6
In view of these reports, we determined the cellular
localization of MAP kinase in megakaryocytes, with a particular
attention to the platelet-yielding demarcation membranes.7
As a control, we determined the localization of another TPO-upregulated
serine/threonine kinase, Mst1.8 Our current Electron
Microscopic (EM) studies, performed as we described
before,9 reveal that a significant fraction of MAP kinase,
but none of Mst1, localizes to the demarcation membranes. In resting
platelets, MAP kinase is found in a cluster pattern associated with the
plasma membrane (which is believed to originate from the megakaryocytic
demarcation membranes),7 as well as over electron-dense
cytoplasmic domains (Figure). In view of
our finding that MAP kinase is localized to the demarcation membranes
in megakaryocytes, it is of interest to note that this kinase was also
shown to be localized in Golgi membranes and involved in their
fragmentation during mitosis in a microtubule-dependent manner.10 Platelet fragmentation likely involves the
cytoskeleton, and perhaps MAP kinases are also involved in this
process.
View larger version (114134135K):
[in this window]
[in a new window]
|
Immunogold Electron Microscopic assays (performed as described
before)9.
(A) Ultrathin frozen sections of rat platelets immunolabeled with
antibodies to ERK (magnification ×45 000). (B) Well-developed
demarcation membranes in a rat (Wistar), TPO-treated
megakaryocyte7,9 immunostained with an antibody to Mst1
(magnification ×48 000), or (C) with an antibody to ERK, which
recognizes ERK1 and ERK2 (magnification ×65 400). The arrows
point to the demarcation membranes. The Mst1 antiserum (gift of Dr
Jonathan Chernoff, Fox Chase Cancer Center, Philadelphia, PA) and
anti-ERK2 (K-23) (Santa Cruz Biotechnology, Santa Cruz, CA) or TR2,
anti-ERK (gift of Michael Weber, University of Virginia Medical School)
were diluted 1:10.
|
|
| |
Acknowledgments |
We thank Dr. Paula Stenberg at the Oregon Health Sciences
University for skilful EM analyses. This work was supported by NIHBI grants HL53080 and HL58547 to K.R. K.R. is an established investigator with the American Heart Association.
Shishinn Sun
Department of Biochemistry, Boston University School of
Medicine, Boston, MA 02118
Carl W. Jackson
Division of Experimental Hematology, St. Jude Children's
Research Hospital, Memphis, TN 38105
Katya Ravid
Department of Biochemistry, Boston University School of
Medicine, Boston, MA 02118
| |
References |
1.
Kaushansky K.
Thrombopoietin: the primary regulator of platelet production.
Blood.
1995;86:419-431[Free Full Text].
2.
Rouyez MC, Boucheron C, Gisselbrecht S, Dusanter-Fourt I, Porteu F.
Control of thrombopoietin-induced megakaryocytic differentiation by the mitogen-activated protein kinase pathway.
Molecular and Cellular Biology.
1997;17:4991-5000[Abstract].
3.
Ray LB, Sturgill TW.
Insulin-stimulated microtubule associated protein kinase is detectable by analytical gel chromatography as a 35-kDa protein in myocytes, adipocytes, and hepatocytes.
Arch Biochem Biophys.
1988;262:307-313[Medline]
[Order article via Infotrieve].
4.
Goedert M, Cohen ES, Jakes R, Cohen P.
p42 MAP kinase phosphorylation sites in microtubule-associated protein tau are dephosphorylated by protein phosphatase 2A1. Implications for Alzheimer's disease.
FEBS Lett.
1992;312:95-99[Medline]
[Order article via Infotrieve].
5.
Olsen MK, Reszka AA, Abraham I.
KT5720 and U-98017 inhibit MAPK and alter the cytoskeleton and cell morphology.
J Cell Physiol.
1998;176:525-536[Medline]
[Order article via Infotrieve].
6.
Lecine P, Italiano JE, Aksoy E, Shivdasani RA.
Molecular analysis of genetically thrombocytopenic mice reveals a potential biochemical pathway for terminal megakaryocyte differentiation and platelet release [abstract].
Blood.
1988;92:569a.
7.
Behnke O.
An electron microscope study of the megacaryocyte of the rat bone marrow. I. The development of the demarcation membrane system and the platelet surface coat.
J Ultrastruct Res.
1968;24:412-433[Medline]
[Order article via Infotrieve].
8.
Sun S, Ravid K.
The role of a serine/threonie protein kinase, Mst1, in megakaryocyte differentiation.
J Cell Biochem.
1999;76:44-48[Medline]
[Order article via Infotrieve].
9.
Zimmet JM, Ladd D, Jackson CW, Stenberg PE, Ravid K.
A role for cyclin D3 in the endomitotic cell cycle.
Mol Cell Biol.
1997;17:7248-59[Abstract].
10.
Acharya U, Mallabiabarrena A, Acgarya JK, Malhortra V.
Signaling via mitogen-activated protein kinase kinase (MEK1) is required for Golgi fragmentation during mitosis.
Cell.
1998;92:183-192[Medline]
[Order article via Infotrieve].
