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Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins
Linköping University, Department of Molecular and Clinical Medicine, Medical Microbiology. Linköping University, Faculty of Health Sciences.
Institute of Biochemistry, Vilnius, Lithuanuia.
Institute of Biochemistry, Vilnius, Lithuanuia.
Institute of Biochemistry, Vilnius, Lithuanuia.
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2001 (English)In: BioTechniques, ISSN 0736-6205, E-ISSN 1940-9818, Vol. 31, no 3, 508-517 p.Article in journal (Refereed) Published
Abstract [en]

Phosphotyrosine signaling plays a vital role in cell regulation - from receptor activation, through stimulation of signal networks and nuclear targeting, to final cellular responses. Here, we propose a new approach to monitor the spatial and temporal aspects of tyrosine phosphorylation and dephosphorylation. The method can be used to determine whether protein tyrosine phosphorylations and dephosphorylations occur in the cytosol or the nucleus and to ascertain whether such modifications are associated with nuclear traffic. Promyelocytic leukemia (HL-60) cells are used as the experimental model. Biotinylated cytosolic proteins from donor cells are used to trace nuclear transport in permeabilized recipient cells. Thereafter, 2-D gel electrophoresis is applied to fractionate the cytosolic and nuclear proteins of the recipient cells, which are subsequently blotted onto polyvinylidene difluoride membranes. The membranes are developed with streptavidin and then reprobed with anti-phosphotyrosine antibodies. The major advantages of the protocol are that it is simple to perform, and reproducible results are obtained by overlaying the patterns of biotinylated and/or tyrosine-phosphorylated proteins. Moreover, several hundred cytosolic and nuclear proteins can be analyzed in parallel. Thus, by comparing the 2-D gel electrophoresis maps of biotinylated and tyrosine-phosphorylated proteins, it is possible to determine the involvement of trafficking of the latter proteins in cell signaling.

Place, publisher, year, edition, pages
2001. Vol. 31, no 3, 508-517 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26005PubMedID: 11570494Local ID: 10457OAI: oai:DiVA.org:liu-26005DiVA: diva2:246553
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
In thesis
1. New aspects on retinoic acid induced HL-60 granulocytic differentiation
Open this publication in new window or tab >>New aspects on retinoic acid induced HL-60 granulocytic differentiation
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The underlying mechanisms of myeloid cell differentiation are still not entirely elucidated. It is therefore an urgent task to extent the basis for a comprehensive model of granulocytes differentiation. A primary goal for studies has been the identification of differentiation markers. These often correspond to genes that are expressed at defined steps during the differentiation process or in the fully differentiated phenotype. Here is demonstrated that the program of phenotype formation entails dramatic changes in long-term tyrosine phosphorylation of some cytoplasmic proteins during retinoic acid (RA)-induced HL-60 cell differentiation to granulocytes. A significant increase in the phosphotyrosine content of a kinase (Erk2) in the commitment and terminal stages of HL-60 cell differentiation (48-120 h) was related to promotion of cell survival and triggering of apoptosis. In addition, some cytosolic proteins of apoptotic origin were delineated in the differentiating population. The pattern of tyrosine phosphorylations of specific cytosolic proteins in maturing and apoptotic granulocytes should be a powerful tool for further elucidation of differentiation mechanisms. We developed a new approach based on cytosolic protein tagging and nuclear import analysis combined with assessment of specific protein tyrosine phosphorylations by two-dimensional gel electrophoresis. Many of the genes involved in the differentiation of multipotent stem cells to specific cellular lineages are still unknown. Accordingly, we searched for additional tyrosine phosphorylated, RA-induced transcription regulators that are translocated into the nucleus during commitment of HL-60 cells to become granulocyte-like cells. It is shown, that transcription factors required for lineage commitment of differentiating (C/EBPß, c-myb) and for survival of differentiated cells (STATs, NFKB) co-operatively promoted signaling in the HL-60 cell line in response to RA. Identification of the tyrosine-phosphorylated proteins being translocated into the nucleus during HL-60 cell differentiation was our further goaL This way, we show for the first time that human gamma~dystrobrevin is expressed in promyelocytic HL-60 cell line. We detected gamma-dystrobrevin in the cytosol and the nuclei of these cells, and, in the latter location; it was modified by tyrosine phosphorylation soon after the onset of the RA-triggered differentiation. Varying patterns of distribution of gamma-dystrobrevin could be discerned in HL-60 promyelocytes, RA-differentiated mature granulocytes, and hwnan neutrophils. Moreover, we found gamma-dystrobrevin in association with actin and myosin light chain. Our results provide new information about potential involvement of gamma-dystrobrevin in signal transduction in myeloid cells during induction of granulocytic differentiation and/or at the commitment stage of differentiation or phagocytic cells. In addition, our study confirms and extends knowledge about the genes involved in granulocytic differentiation and growth arrest. This shuiy corroborates the value of cDNA RDA to isolate factors involved in myeloid maturation. In conclusion, the more detailed analysis of granulocytic differentiation of HL-60 cells presented here provides new data that should allow further refinement of models of myeloid differentiation.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2002. 119 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 723
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26659 (URN)11225 (Local ID)91-7373-165-X (ISBN)11225 (Archive number)11225 (OAI)
Public defence
2002-04-11, Berzeliussalen, Universitetssjukhuset, Linköping, 09:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-09-17Bibliographically approved

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