The NGFI-B protein, an inducible member of the thyroid/steroid receptor family, is rapidly modified posttranslationally.
The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of congruent to 61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.
Fahrner
T J
TJ
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Carroll
S L
SL
Milbrandt
J
J
D000310:Adrenal Gland Neoplasms; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005810:Multigene Family; D009414:Nerve Growth Factors / Q000494:pharmacology; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D010673:Pheochromocytoma; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid / Q000235:genetics*; D011988:Receptors, Thyroid Hormone / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000097:blood / Q000235:genetics* / Q000302:isolation & purification
D004268:DNA-Binding Proteins; D009414:Nerve Growth Factors; C539820:Nr4a1 protein, rat; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D018160:Receptors, Cytoplasmic and Nuclear; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; D014157:Transcription Factors; D016222:Fibroblast Growth Factor 2; D000001:Calcimycin; D004815:Epidermal Growth Factor; D013755:Tetradecanoylphorbol Acetate