The zinc finger protein NGFI-A exists in both nuclear and cytoplasmic forms in nerve growth factor-stimulated PC12 cells.
The NGFI-A gene, which encodes a zinc finger protein with a predicted molecular mass of congruent to 54 kDa, is rapidly activated in PC12 cells by nerve growth factor (NGF). As a transcription factor, NGFI-A is a potentially important mediator of the cellular response to this growth factor. To characterize this protein, antibodies to four different domains of NGFI-A were raised. Immunogens included a bacterial trpE/NGFI-A fusion protein (A310) and three peptides predicted from the NGFI-A cDNA nucleotide sequence. Three of these antisera recognize two predominant NGFI-A species in NGF-stimulated PC12 cells: a 54-kDa form and a closely spaced doublet at 84 kDa. However, one of these antisera, directed against the last 15 carboxyl terminal residues of NGFI-A, recognizes only the 84-kDa species. Both NGFI-A species are rapidly induced in PC12 cells by NGF, phorbol ester, and the calcium ionophore A23187. Pulse-chase analysis revealed no obvious precursor-product relationship between these two forms and demonstrated that both the 54- and 84-kDa species are short-lived proteins. V8 protease digestion of the 54- and 84-kDa forms resulted in the formation of several small peptides that were common to both species. The digest of each species also contained one large, relatively V8 protease-resistant fragment that was substantially larger in digests of the 84-kDa form. These two fragments contained common epitopes and were derived from the amino-terminal portion of the NGFI-A protein. When NGFI-A was incubated with alkaline phosphatase, the 84-kDa doublet resolved into a single band with slightly increased mobility, indicating that this form is phosphorylated. Cell fractionation studies demonstrated that the 84-kDa species are found exclusively in the nucleus, while the 54-kDa form resides solely in the cytoplasm. It therefore appears that the 54-kDa form lacks the signal necessary for nuclear localization and is missing a portion of the carboxyl terminal domain.
Day
M L
ML
Department of Pathology, Washington University Medical School, St. Louis, Missouri 63110.
Fahrner
T J
TJ
Aykent
S
S
Milbrandt
J
J
D000310:Adrenal Gland Neoplasms; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification; D007106:Immune Sera; D008667:Metalloproteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000235:genetics* / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis; D010673:Pheochromocytoma; D014176:Protein Biosynthesis; D051381:Rats; D014158:Transcription, Genetic; D015032:Zinc / Q000378:metabolism*
D004268:DNA-Binding Proteins; D007106:Immune Sera; D008667:Metalloproteins; D009414:Nerve Growth Factors; D010455:Peptides; D015032:Zinc