Identification of a zinc finger protein whose subcellular distribution is regulated by serum and nerve growth factor

Abstract

A subclass of zinc finger proteins containing a unique protein motif called the positive regulatory (PR) domain has been described. The members include the PRDI-BF1/Blimp-1 protein, the Caenorhabditis elegans egl-43 and EVI1 gene products, and the retinoblastoma interacting protein RIZ. Here we describe a member of this family, SC-1, that exhibits several distinctive features. First, SC-1 interacts with the p75 neurotrophin receptor and is redistributed from the cytoplasm to the nucleus after nerve growth factor (NGF) treatment of transfected COS cells. The translocation of SC-1 to the nucleus was specific for p75, as NGF binding to the TrkA receptor did not lead to nuclear localization of SC-1. Thus, SC-1 provides a downstream transducer for the effects of NGF through the p75 neurotrophin receptor. Under normal growth conditions, SC-1 was found predominantly in the cytoplasm. On serum-starvation, SC-1 also translocated into the nucleus. A direct correlation between nuclear expression of SC-1 with the loss of BrdUrd incorporation was observed. These results imply that SC-1 may be involved in events associated with growth arrest.

Figure 1

Sequence and RNA analysis of SC-1. (A) Full-length amino acid sequence of the rat SC-1. The PR domain is indicated in italics. The zinc fingers are underlined. (B) Schematic representation of the SC-1 amino acid sequence. Six zinc fingers are located at the COOH terminus. Two serine-rich regions are indicated by steepled boxes, a proline-rich region is indicated as the squared box, and the PR domain is indicated. Below, a schematic representation of the original yeast clone coding sequence is shown for comparison containing amino acids 490–814 (truncated SC1). (C) Northern blot of SC-1 mRNA in adult rat tissues (20 μg total RNA). The blot was stained with methylene blue to visualize the amount of RNA loaded onto each lane (Lower). (D) SC-1 and p75 receptor mRNA expression in cultured Schwann cells and PC12 cells (10 μg total RNA each lane). Lanes: 1, proliferating Schwann cells; 2, quiescent Schwann cells; 3, PC12 cells. (E) An alignment of the PR domains from SC-1, Blimp, RIZ, evi1, and egl-43. The identical amino acids are boxed and shaded. Dashes indicate sequence gaps.

Figure 1

Sequence and RNA analysis of SC-1. (A) Full-length amino acid sequence of the rat SC-1. The PR domain is indicated in italics. The zinc fingers are underlined. (B) Schematic representation of the SC-1 amino acid sequence. Six zinc fingers are located at the COOH terminus. Two serine-rich regions are indicated by steepled boxes, a proline-rich region is indicated as the squared box, and the PR domain is indicated. Below, a schematic representation of the original yeast clone coding sequence is shown for comparison containing amino acids 490–814 (truncated SC1). (C) Northern blot of SC-1 mRNA in adult rat tissues (20 μg total RNA). The blot was stained with methylene blue to visualize the amount of RNA loaded onto each lane (Lower). (D) SC-1 and p75 receptor mRNA expression in cultured Schwann cells and PC12 cells (10 μg total RNA each lane). Lanes: 1, proliferating Schwann cells; 2, quiescent Schwann cells; 3, PC12 cells. (E) An alignment of the PR domains from SC-1, Blimp, RIZ, evi1, and egl-43. The identical amino acids are boxed and shaded. Dashes indicate sequence gaps.

Figure 2

Interaction of p75 and SC-1. (A) Transfected 293 cells expressing the rat p75 cDNA; an unrelated protein, Flag-tagged BRE, and a Flag-tagged truncated SC-1 cDNA (pSC2; amino acids 490–814; Fig. 1B) were subjected to immunoprecipitation using anti-Flag antibodies, followed by blotting with the anti-p75 antibody, 9992. The arrowheads demonstrate the position of p75 on the blot, which migrated as a doublet, representing products of differential glycosylation of p75. Lanes: 1, p75 and pSC2; 2, p75 and Flag vector; 3, p75 and an unrelated protein Flag-Bre cDNA (22); 4, 293 cells transfected with p75 cDNA alone, probed with anti-p75, 9992 antibody. In the lower panel, the transfected proteins were detected after immunoprecipitation and Western blotting with anti-Flag antibodies. (B) SC-1 interacts with the juxtamembrane domain of human p75. GSTp75 fusion proteins were used for assessing the binding of in vitro translated SC-1 to p75 intracellular sequences. The binding reaction was carried out by using 3 μg of the GSTp75 fusion proteins on glutathione beads. The left-most lane represents input in vitro translated SC-1 protein. The proteins were resolved on a 7% SDS/PAGE gel. The arrowhead indicates the migration of the major in vitro translated product of SC-1.

Figure 3

Subcellular localization of SC-1 is regulated by both serum and NGF. Flag-tagged SC-1 (pSC1) was either transfected alone or in combination with either p75 or TrkA into COS cells. Transfected cells were stained with anti-Flag antibodies and appropriate receptor antibodies followed by the secondary antibodies coupled to FITC for SC-1 or rhodamine for p75 or TrkA staining. (Upper) COS cells transfected with SC-1 in 10% fetal calf serum and after 1 hour of serum withdrawal. (Lower) COS cells were transfected with SC-1 and p75 cDNA or with SC-1 and TrkA cDNAs. Immunostaining for Flag-SC-1 was carried out before and after 1 hour of treatment with 100 ng/ml NGF. αp75 and αTrkA panels represent expression p75 and TrkA receptors after staining with 9651 (11) or RTA antibodies (12).

Figure 4

Quantitation of the subcellular distribution of SC-1. Shown is quantitation of the effects of serum starvation and NGF treatment on the subcellular distribution of SC-1 in transfected COS cells. The total number of cells displaying SC-1 expression was measured and is represented as a percentage (%). Three different states were monitored: nuclear, cytoplasmic, or intermediate staining. Statistical analysis was carried out by using the χ² test. The P values obtained varied between P < 0.001 and P < 0.01.

Figure 5

Incorporation of BrdUrd after transfection of COS-1 cells with SC-1. Expression of SC-1 was detected by using anti-Flag antibody (Kodak) and by FITC-coupled secondary antibody. Incorporation of BrdUrd in COS cells was detected by using an anti-BrdUrd primary antibody and a Texas-Red-coupled secondary antibody. (A) COS cells stained for BrdUrd. (B) COS cells stained for Flag-SC-1. (C) An overlay of A and B showing that the BrdUrd and SC-1 immunoreactivities are mutually exclusive. (D) Quantitation of BrdUrd-positive COS cells transfected with SC-1.