Nuclear matrix bound fibroblast growth factor receptor is associated with splicing factor rich and transcriptionally active nuclear speckles.
We have used confocal microscopy combined with computer image analysis to evaluate the functional significance of a constitutively expressed form of the receptor tyrosine kinase FGFR1 (fibroblast growth factor receptor 1) in the nucleus of rapidly proliferating serum stimulated TE 671 cells, a medullobastoma human cell line. Our results demonstrate a limited number of large sites and numerous smaller sites of FGFR1 in the nuclear interior. The larger sites showed virtually complete colocalization (>90%) with splicing factor rich nuclear speckles while the smaller sites showed very limited overlap (<20%). Similar results were found for several other proliferating cell lines grown in culture. An in situ transcription assay was used to determine colocalization with transcription sites by incorporating 5-bromouridine triphosphate (BrUTP) followed by dual staining for BrUTP and FGFR1. These results combined with those from using an antibody against the large subunit of RNA polymerase II suggest a significant degree of colocalization (26-38%) over both the large and small sites. No colocalization was detected with sites of DNA replication. The spatial arrangements of FGFR1 sites and colocalization with nuclear speckles were maintained following extraction for nuclear matrix. Moreover, immunoblots indicated a significant enrichment of FGFR1 in the nuclear matrix fraction. Our findings suggest an involvement of a nuclear matrix bound FGFR1 in transcriptional and RNA processing events in the cell nucleus. We further propose that nuclear speckles, aside from a role in transcriptional/RNA processing events, may serve as fundamental regulatory factories for the integration of diverse signaling and regulatory factors that impact transcription and cellular regulation.
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, New York 14260, USA.
Suryanarayan
S
Somanathan
Ewa K
EK
Stachowiak
Alan J
AJ
Siegel
Michal K
MK
Stachowiak
Ronald
R
Berezney
D000818:Animals; D002460:Cell Line; D022003:Cell Nucleus Structures / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D018613:Microscopy, Confocal; D015530:Nuclear Matrix / Q000378:metabolism*; D009698:Nucleoproteins / Q000378:metabolism*; D021381:Protein Transport; D012319:RNA Polymerase II / Q000378:metabolism; D012326:RNA Splicing; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions; D014158:Transcription, Genetic
D009698:Nucleoproteins; D017468:Receptors, Fibroblast Growth Factor; D012319:RNA Polymerase II