Mechanisms Regulating the Association of Protein Phosphatase 1 with Spinophilin and Neurabin

Protein phosphorylation is a key mediator of signal transduction, allowing for dynamic regulation of substrate activity. Whereas protein kinases obtain substrate specificity by targeting specific amino acid sequences, serine/threonine phosphatase catalytic subunits are much more promiscuous in their ability to dephosphorylate substrates. To obtain substrate specificity, serine/threonine phosphatases utilize targeting proteins to regulate phosphatase subcellular localization and catalytic activity. Spinophilin and its homologue neurabin are two of the most abundant dendritic spine-localized protein phosphatase 1 (PP1) targeting proteins. The association between spinophilin and PP1 is increased in the striatum of animal models of Parkinson's disease (PD). However, mechanisms that regulate the association of spinophilin and neurabin with PP1 are unclear. Here, we report that the association between spinophilin and PP1α or PP1γ1 was increased by CDK5 expression and activation in a heterologous cell system. This increased association is at least partially due to phosphorylation of PP1. Conversely, CDK5 expression and activation decreased the association of PP1 with neurabin. As with dopamine depletion, methamphetamine (METH) abuse causes persistent alterations in dopamine signaling which influence striatal medium spiny neuron function and biochemistry. Moreover, both METH toxicity and dopamine depletion are associated with deficits in motor control and motor learning. Pathologically, we observed a decreased association of spinophilin with PP1 in rat striatum evaluated one month following a binge METH paradigm. Behaviorally, we found that loss of spinophilin recapitulates rotarod pathology previously observed in dopamine-depleted and METH-treated animals. Together, these data have implications in multiple disease states associated with altered dopamine signaling such as PD and psychostimulant drug abuse and delineate a novel mechanism by which PP1 interactions with spinophilin and neurabin may be differentially regulated

The association of spinophilin with disks large-associated protein 3 (SAPAP3) is regulated by metabotropic glutamate receptor (mGluR) 5

Spinophilin is the most abundant protein phosphatase 1 targeting protein in the postsynaptic density of dendritic spines. Spinophilin associates with myriad synaptic proteins to regulate normal synaptic communication; however, the full complement of spinophilin interacting proteins and mechanisms regulating spinophilin interactions are unclear. Here we validate an association between spinophilin and the scaffolding protein, disks large-associated protein 3 (SAP90/PSD-95 associated protein 3; SAPAP3). Loss of SAPAP3 leads to obsessive-compulsive disorder (OCD)-like behaviors due to alterations in metabotropic glutamate receptor (mGluR) signaling. Here we report that spinophilin associates with SAPAP3 in the brain and in a heterologous cell system. Moreover, we have found that expression or activation of group I mGluRs along with activation of the mGluR-dependent kinase, protein kinase C β, enhances this interaction. Functionally, global loss of spinophilin attenuates amphetamine-induced hyperlocomotion, a striatal behavior associated with dopamine dysregulation and OCD. Together, these data delineate a novel link between mGluR signaling, spinophilin, and SAPAP3 in striatal pathophysiology

Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression

BACKGROUND: TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression. METHODS: DNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry. RESULTS: Normal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL. CONCLUSION: Loss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression

TRAIL mRNA levels are markedly higher in normal oral epithelial cells (NOM) in comparison to immortalized (16B), premalignant (L1 and L2) and malignant oral epithelial cells (16BTu, 1386Tu, 686Tu, 101A, 1386Ln and 686Ln)

<p><b>Copyright information:</b></p><p>Taken from "Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression"</p><p>http://www.biomedcentral.com/1471-2407/7/108</p><p>BMC Cancer 2007;7():108-108.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1924860.</p><p></p> Relative RNA expression levels of TRAIL were determined using Affymetrix U133A oligonucleotide microarrays. Signal intensity for TRAIL was calculated using the Affymetrix MAS 5.0 probe level algorithm. : Normal oral epithelial cells; : Normal oral epithelial cells immortalized by HPV-16 transfection (HOK-16B). : OPM cell line MSK-Leuk1; : OPM cell line MSK-Leuk2; : tumorigenic cell line derived from HOK-16B; (HOK-16B-Bap-T); : Cell lines derived from primary OSCC. : Cell lines derived from synchronous lymph node metastases of 1386Tu and 686Tu tumors

Immunohistochemical staining for cleaved caspase 3 to detect apoptotic cells among tumor cells and tumor-infiltrating lymphocytes

<p><b>Copyright information:</b></p><p>Taken from "Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression"</p><p>http://www.biomedcentral.com/1471-2407/7/108</p><p>BMC Cancer 2007;7():108-108.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1924860.</p><p></p> Examples showing the detection apoptotic cells among tumor cells (LI-Cas-Tu)) and TIL (LI-Cas-TIL) based on their immunoreactivity for cleaved caspase-3 (× 400)

Loss of TRAIL expression in oral leukoplakia with moderate epithelial dysplasia

<p><b>Copyright information:</b></p><p>Taken from "Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression"</p><p>http://www.biomedcentral.com/1471-2407/7/108</p><p>BMC Cancer 2007;7():108-108.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1924860.</p><p></p> A: Hematoxylin and eosin staining; B: Immunohistochemical staining for TRAIL (× 100). Note that immunoreactivity for TRAIL is significantly reduced in dysplasia (arrows) compared to adjacent uninvolved mucosa (N)

Loss of TRAIL expression pattern in oral leukoplakia with severe dysplasia

<p><b>Copyright information:</b></p><p>Taken from "Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression"</p><p>http://www.biomedcentral.com/1471-2407/7/108</p><p>BMC Cancer 2007;7():108-108.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1924860.</p><p></p> A: Hematoxylin and eosin staining (× 100); B: Immunohistochemical staining for TRAIL (× 100). Note that TRAIL expression is mostly lost in dysplastic epithelium (arrow). In contrast, mononuclear immune cell infiltrate (MNC) associated with dysplasia reveals intense staining for TRAIL

The expression levels of TRAIL and DcR2 are markedly reduced in oral premalignancy compared to normal oral mucosa

<p><b>Copyright information:</b></p><p>Taken from "Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression"</p><p>http://www.biomedcentral.com/1471-2407/7/108</p><p>BMC Cancer 2007;7():108-108.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1924860.</p><p></p> Graphic depiction of the mean expression levels of TRAIL and TRAIL-R in patient matched normal oral mucosa (NOM), oral premalignancies (OPM: leukoplakias with dysplasia). Mean expression levels of DR4, DR5 and DcR1 did not differ significantly between normal oral mucosa (NOM) and oral premalignancies (OPM)

TRAIL protein is detectable only in normal oral epithelial cells (lanes 1 and 2) but not in premalignant (lane 3) and malignant oral epithelial cell lines (lanes 4 and 5)

<p><b>Copyright information:</b></p><p>Taken from "Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression"</p><p>http://www.biomedcentral.com/1471-2407/7/108</p><p>BMC Cancer 2007;7():108-108.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1924860.</p><p></p> Total cellular proteins were extracted from two normal oral epithelial samples (NOM-1 and 2-lanes 1 and 2), OPM cell line (MSK-Leuk1-lane 3), primary OSCC cell line (1386Tu-lane 4) and metastatic OSCC cell line (1386Ln-lane 5) and analyzed for TRAIL protein by Western blotting. : none specific bands

Expression patterns of TRAIL-R, DR4, DR5, DcR1 and DcR2 are not different between primary and synchronous metastatic OSCC

<p><b>Copyright information:</b></p><p>Taken from "Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression"</p><p>http://www.biomedcentral.com/1471-2407/7/108</p><p>BMC Cancer 2007;7():108-108.</p><p>Published online 25 Jun 2007</p><p>PMCID:PMC1924860.</p><p></p> Immunohistochemical expression patterns of TRAIL receptors in primary (Iry) and lymph node metastasis (Met.) of OSCC from the same patient. (× 200)