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, 16 (24), 7821-31

STEP61: A Member of a Family of Brain-Enriched PTPs Is Localized to the Endoplasmic Reticulum

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STEP61: A Member of a Family of Brain-Enriched PTPs Is Localized to the Endoplasmic Reticulum

A Bult et al. J Neurosci.

Abstract

The STEP family of protein tyrosine phosphatases is highly enriched within the CNS. Members of this family are alternatively spliced to produce both transmembrane and cytosolic variants. This manuscript describes the distinctive intracellular distribution and enzymatic activity of the membrane-associated isoform STEP61. Transfection experiments in fibroblasts, as well as subcellular fractionations, sucrose density gradients, immunocytochemical labeling, and electron microscopy in brain tissue, show that STEP61 is an intrinsic membrane protein of striatal neurons and is associated with the endoplasmic reticulum. In addition, structural analysis of the novel N-terminal region of STEP61 reveals several motifs not present in the cytosolic variant STEP46. These include two putative transmembrane domains, two sequences rich in Pro, Glu, Asp, Ser, and Thr (PEST sequences), and two polyproline-rich domains. Like STEP46, STEP61 is enriched in the brain, but the recombinant protein has less enzymatic activity than STEP46. Because STEP46 is contained in its entirety within STEP61 and differs only in the extended N terminus of STEP61, this amino acid sequence is responsible for the association of STEP61 with membrane compartments and may also regulate its enzymatic activity.

Figures

Fig. 1.
Fig. 1.
STEP61 encodes a PTP with two transmembrane, two PEST sequences, and two potential SH3 binding sites.A, The two hydrophobic domains are indicated by thedouble underline, and the two polyproline-rich domains are indicated by a single underline. Two PEST sequences are enclosed in brackets, and the phosphatase domain is shown in bold. Five conservative amino acid changes from the original rat STEP46 sequence are indicated byasterisks and reflect the expected variations of sequence between mouse and rat. STEP46 sequence begins at methionine residue 173 (indicated by a vertical bar).B, Hydrophilicity analysis of the STEP61-predicted amino acid sequence indicates two stretches of hydrophobic amino acids of 20 and 23 amino acids are present at the N terminus. The plot was obtained using the MacVector sequence analysis software and a Kyte–Doolittle algorithm with a window of seven amino acids. C, Schematic representation of STEP61 and comparison with the cytosolic STEP variant STEP46. The transmembrane domains (TM), PEST sequences, and polyproline domains (PP) are shown.
Fig. 2.
Fig. 2.
STEP61 mRNA is enriched in the CNS. Poly(A+)-selected mRNA (2 μg) from the indicated mouse organs was loaded onto a denaturing gel, electrophoresed, and transferred to nylon membrane. A STEP61-specific probe was generated by PCR and randomly primed with 32P. After 18 hr of hybridization, the blot was washed and exposed with intensifying screens overnight at −80°C.
Fig. 3.
Fig. 3.
STEP61 in brain: biochemical analysis.A, Immunoblots of subcellular fractionation of rat brain. Equivalent amounts of protein from each fraction (50 μg) were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with the STEP61 monoclonal antibody 23E5 and with a polyclonal antibody against synaptophysin (p38). H, Total homogenate; P1, consisting primarily of unbroken cells and nuclei; P2, plasma membrane and larger organelles;P3, membranes of smaller organelles; S3, cytosol; LP1, pellet after hypotonic lysis of synaptosomes and contains larger organelles within synaptosomes;LP2, high-spin pellet of LS1 supernatant and containing microsomal fraction within synaptosomes; LS2, supernatant of LP2 spin. MW standards are shown on theleft. B, Thrombin digestion of STEP61-fusion protein. The released recombinant STEP polypeptide (lane 1) was compared with the mobilities of endogenous STEP proteins enriched in P3 fraction (lane 2). C, N-glycosidase F treatment of P3 membranes. Samples were processed at 37°C overnight in the presence (+) or absence (−) of enzyme. Blots were processed with antibodies against STEP (top) or against the control synaptophysin (p38, bottom). D, Distribution of STEP61 after sucrose density gradient fractionation and comparison with ER and non-ER-associated proteins. Membrane fractions enriched for the higher MW STEP-immunoreactive peptides (P3 and LP2) were applied to continuous sucrose gradients (0.4–2 m). After ultracentrifugation, equal volumes from each of 12 aliquots were loaded onto 10% SDS polyacrylamide gels and processed in parallel with antibodies against calnexin, STEP, and synaptophysin (p38), as indicated on the right. MW markers are shown on the left.
Fig. 4.
Fig. 4.
STEP61 is an integral membrane protein. Membrane fractions (P3) were washed in different buffer conditions as indicated and spun, and pellets (P) and supernatants (S) were collected. Protein (∼50 μg) from each fraction was subjected to SDS-PAGE, transferred to nitrocellulose, and probed with the monoclonal antibody 23E5. The majority of STEP isoforms remain membrane-associated until washed in 2% Triton X-100.
Fig. 5.
Fig. 5.
STEP61 immunoreactivity is localized in the perinuclear region. A, Immunoperoxidase labeling of cortical neurons with Nod antisera showed that the perinuclear region of pyramidal neurons was labeled. B, Preabsorption of Nod antisera with STEP61 fusion protein eliminated all immunofluorescent staining in control sections.CF, To determine whether STEP immunoreactivity was associated with the ER, immunofluorescent double-labeling studies were performed with the Nod antisera (C, E) and an antibody specific for PDI, a resident protein of the ER (D,F). In cortical neurons (C,D), STEP colocalized with PDI in proximal dendrites and surrounding the nucleus. A similar pattern was also seen in hippocampal neurons (E, F). Scale bar, 10 μm.
Fig. 6.
Fig. 6.
Electron microscopy demonstrates localization of STEP61 to ER. In sections incubated in Nod antibody against STEP61, a high density of gold particles was evident over free ribosomes (A) and the rough ER (B). Preabsorption of STEP antisera with STEP peptide-abolished staining (C). Specificity of staining was also shown by labeling sections with synaptophysin antibodies, which failed to label rough ER (D) but gave strong labeling of synaptic vesicles in terminal boutons (F). By contrast, STEP immunogold staining was not observed over more distal processes, including axons and synaptic boutons (E). InA and D, arrows indicate nuclear envelope. In A and C,boxes highlight some gold particles to distinguish them from ribosomes. In B, arrowheads show labeled rough ER. Scale bar, 0.5 μm.
Fig. 7.
Fig. 7.
Transfection of STEP61 into fibroblasts shows a reticular staining pattern relative to the cytosolic STEP46 variant. STEP61 and STEP46 cDNAs were transiently transfected into CHO cells and detected by immunohistochemistry using the monoclonal antibody 23E5 followed by rhodamine-conjugated goat anti-mouse IgG. In transfected cells, STEP61 presented the characteristic reticular distribution of proteins associated with the ER (top). Note the perinuclear accumulation of STEP61. In contrast, STEP46 immunoreactivity was evenly distributed in the cytoplasm of transfected cells, consistent with STEP46being a soluble cytosolic protein (bottom). Scale bar, 22 μm.
Fig. 8.
Fig. 8.
STEP61 is less active than the cytosolic variant STEP46. The phosphatase activity of the recombinant proteins STEP61 and STEP46 was compared. STEP61 had approximately sixfold less phosphatase activity than STEP46. STEP61-GST and STEP46-GST fusion proteins were affinity-purified on glutathione–agarose beads after induction by IPTG and assayed for phosphatase activity in the presence of 1% Triton-X 100 using the substrate pNPP. The values of STEP61 and STEP46 represent the mean ± SE of two separate assays. GST fusion protein alone (GST-alone) is the negative control.

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