Parallel assessment of tyrosine phosphorylation and nuclear targeting of proteins.
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-phosphor lated proteins, it is possible to determine the involvement of trafficking of the latter proteins in cell signaling.
Kulyte
A
A
Institute of Biochemistry, Vilnius, Lithuania. agnku@ihm.liu.se
Navakauskiene
R
R
Treigyte
G
G
Gineitis
A
A
Magnusson
K E
KE
D001692:Biological Transport; D019921:Biotinylation; D002463:Cell Membrane Permeability; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D018922:HL-60 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis / Q000302:isolation & purification; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism*; D011145:Polyvinyls; D005465:Fluorocarbon Polymers
D004072:Digitonin; D009687:Nuclear Proteins; D019000:Phosphotyrosine; D011145:Polyvinyls; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers