Cellular and molecular characterization of a brain-enriched protein tyrosine phosphatase

Abstract

Regional variations in the expression of a striatal enriched protein tyrosine phosphatase called STEP were studied in the adult rat brain by a combination of immunocytochemistry, lesion studies, Western blotting, and in situ hybridization. Monoclonal antibodies generated against STEP identified multiple polypeptides of M(r) 46, 37, 33 and a doublet of M(r) 64–66 kDa on Western blots. Although the three STEP immunoreactive bands with lower molecular weights were enriched in cytosolic fractions, the 64–66 kDa doublet was enriched in membrane fractions. All of the immunoreactive forms were abundant in the caudate-putamen and were present in lower amounts or were undetectable in other brain regions. In substantia nigra, the M(r) 64–66 kDa doublet was not detected but bands with M(r) 46, 37, and 33 kDa were present. Immunocytochemical and lesion experiments demonstrated that the cytosolic STEP isoforms present in the substantia nigra are in presynaptic axons originating from the projection neurons of the caudate putamen, which innervate this structure. Additional in situ hybridization studies showed that STEP mRNA expression patterns correlate with the patterns of immunocytochemical staining. These findings indicate that there are multiple polypeptide isoforms of STEP enriched in the basal ganglia and related structures which differ in terms of their intracellular locations and functional roles.

The Striatal-Enriched Protein Tyrosine Phosphatase Gates Long-Term Potentiation and Fear Memory in the Lateral Amygdala

“…STEP is localized to brain regions involved in learning and memory, including the striatum, hippocampus, amygdala, nucleus accumbens, and cortex (Lombroso et al, 1993;Boulanger et al, 1995). Alternative splicing of a single STEP gene produces a 46 kDa cytosolic member (STEP 46 ) and a 61 kDa membrane-associated member (STEP 61 ) (Bult et al, 1996).…”

“…These include two transmembrane domains, two polyproline domains, and two PEST sequences. Electron microscopy and biochemical analyses have localized STEP 61 to the endoplasmic reticulum and postsynaptic terminal, where it is associated both with the postsynaptic density (Bult et al, 1996;Boulanger et al, 1995;Oyama et al, 1995) and the N-methyl-D-aspartate (NMDA) receptor protein complex (Pelkey et al, 2002). STEP-family proteins contain an ERK binding domain, the kinase interacting motif (KIM), which is required for the association of STEP with the ERKs (Paul et al, 2003;Pulido et al, 1998).…”

Hypoxia‐Ischemia in Perinatal Rat Brain Induces the Formation of a Low Molecular Weight Isoform of Striatal Enriched Tyrosine Phosphatase (STEP)

“…The various STEP isoforms were identified by immunoblotting with a monoclonal antibody to STEP (23E5) that recognizes all STEP isoforms identified to date (Boulanger et al, 1995). Proteins were resolved by SDS gel electrophoresis on 10% polyacrylamide gels (Laemmli, 1970) and transferred to nitrocellulose membranes as described elsewhere (Gurd, 1992).…”

“…P7 rat brains were analyzed for the presence of STEP by immunoblotting with an antibody that recognizes all known isoforms of STEP (Boulanger et al, 1995). STEP 61 was the major form of STEP in the P7 forebrain (Fig.…”

“…Subsequent studies have identified two subfamilies of STEP proteins: a group of higher molecular weight (M r ) isoforms that are enriched in membrane compartments, and a group of lower M r isoforms, some of which are membrane-associated and others of which are cytosolic proteins (Boulanger et al, 1995;Sharma et al, 1995;Bult et al, 1996Bult et al, , 1997.…”