Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

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Phosphorylation of Caveolin-1 in Bovine Rod Outer Segments in vitro by an Endogenous Tyrosine Kinase

Michael H. Elliott and Abboud J. Ghalayini

1 Introduction

Caveolin-1 (Cav-1), the principal protein component of caveolar membrane domains (Glenney, Jr. and Soppet, 1992; Kurzchalia et al., 1992; Rothberg et al., 1992), was originally identified as a major tyrosine phosphoprotein in Rous sarcoma virus (v-Src) transformed cells (Glenney, Jr. and Zokas, 1989). Although Cav-1 can be phosphorylated on several tyrosine residues (Nomura and Fujimoto, 1999; Schlegel et al., 2001), the most well-characterized phosphorylation site on Cav-1 is tyrosine-14, a site specifically recognized by monoclonal antibodies developed to detect this phosphorylated residue (Lee et al., 2000; Nomura and Fujimoto, 1999). Cav-1 was identified as a phosphoprotein over fifteen years ago, yet surprisingly, little is known about the functional significance of Cav-1 tyrosine phosphorylation. Cav-1 is reportedly phosphorylated by several Src family non-receptor tyrosine kinases (Labrecque et al., 2004; Li et al., 1996; Sanguinetti et al., 2003; Sanguinetti and Mastick, 2003) and is phosphorylated following growth factor, insulin, and estrogen stimulation (Kim et al., 2000; Kiss et al., 2005; Lee et al., 2000; Maggi et al., 2002; Mastick et al., 1995; Orlichenko et al., 2006). Importantly, Cav-1 phosphorylation occurs in response to a variety of cellular stresses (Cao et al., 2004; Chen et al., 2005; Sahasrabuddhe et al., 2006; Sanguinetti et al., 2003; Sanguinetti and Mastick, 2003; Volonte et al., 2001) and has been implicated in the regulation of apoptosis (Shajahan et al., 2006).

Cav-1 is expressed in several retinal cell-types including photoreceptors (BoeszeBattaglia et al., 2002; Elliott et al., 2003; Kachi et al., 2001; Nair et al., 2002; Senin et al., 2004), retinal pigment epithelium (Bridges et al., 2001; Mora et al., 2006), and retinal vascular endothelial cells (Feng et al., 1999; Stitt et al., 2000). The protein is specifically enriched in detergent-resistant membranes derived from rod outer segment (ROS) membranes (Elliott et al., 2003; Martin et al., 2005; Nair et al., 2002;

M.H. Elliott

Department of Ophthalmology, University of Oklahoma Health Sciences Center; Dean A. McGee Eye Institute, Oklahoma City, OK 73104, USA, Tel: 405-271-8316, Fax: 405-271-8128

e-mail: michael-elliott@ouhsc.edu

Senin et al., 2004) and disk preparations (Boesze-Battaglia et al., 2002). Immunocytochemistry suggests that it is more abundant in photoreceptor inner segments (Elliott et al., 2003) and that it is also present in synaptic termini (Kachi et al., 2001; Kim et al., 2006). To date, there is little information regarding Cav-1 phosphorylation in retinal cells and its potential functional significance. In the current study, we investigated the phosphorylation of Cav-1 by an endogenous tyrosine kinase(s) in rod outer segment preparations in vitro.

2 Methods

2.1Preparation of Bovine ROS, in Vitro Phosphorylation and Inhibition by PP2

Bovine ROS were prepared from retinas on continuous sucrose gradients (25–50%) as previously described (Zimmerman and Godchaux III, 1982) with slight modification (Elliott et al., 2003). Tyrosine-phosphorylated ROS were prepared by incubation in phosphorylation buffer [50mM Tris-HCl (pH 7.4), 100 mM NaCl, 2 mM MgCl2, 1 mM ATP, 1 mM Na3VO4] for 30 minutes at 37◦C as previously described (Bell et al., 1999; Bell et al., 2000). Control ROS were incubated in the same buffer without ATP. In some experiments, ROS (1 mg/ml) were incubated (30 min; 37◦C) with or without PP2 (10 M), a specific Src kinase family inhibitor prior to addition of ATP.

2.2Preparation of Detergent-resistant Membranes (DRM) from ROS

Detergent-resistant membranes (DRM) were prepared from bovine ROS using icecold 1% Triton X-100 according to a previously described modification (Elliott et al., 2003; Martin et al., 2005) of the method of Seno et al., (2001). The detergent:phospholipid molar ratio under these conditions is 3:1 (Martin et al., 2005).

2.3 Immunoprecipitation

Immunoprecipitations from solubilized ROS membranes (100–200 g) were carried out as previously described (Ghalayini et al., 2002) except that the buffer was supplemented with 60 mM octylglucoside to efficiently solubilize Cav-1 (Elliott et al., 2003). Antibodies (1–2 g) used for immunoprecipitations were: monoclonal anti-phosphotyrosine (PY99); polyclonal anti-Cav-1 (N-20); polyclonal anti-c-Src (SRC2) and normal rabbit or mouse IgG to control for non-specific binding. All antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Immune complexes were recovered by binding to protein A/G-coupled agarose and were

washed four times with solubilization buffer prior to elution with reducing SDSPAGE sample buffer.

2.4 SDS-PAGE and Western Blot Analysis

Proteins were resolved by reducing SDS-PAGE, transferred to PVDF membranes (0.45 m) and blots were incubated with anti-PY99 (1:500), anti-Cav-1 (1:200), anti-PY14-Cav-1 (1:2500; BD Biosciences, San Jose, CA), or anti-c-Src (1:200) followed by incubation with appropriate HRP-coupled secondary antibody. In some experiments, parallel gels were stained with GelCode Blue staining reagent (Pierce, Rockford, IL) according to the manufacturer’s instructions.

3 Results

To determine whether Cav-1 can be phosphorylated by endogenous kinases in ROS, purified ROS membranes were incubated with ATP, followed by immunoprecipitation with several antibodies. As shown in Fig. 1A, Cav-1 phosphorylated on tyrosine-14, was recovered in immunoprecipitates only from ROS membranes incubated with ATP. Phosphorylated Cav-1 migrated as a cluster of distinct bands that may represent different phosphospecies. In other experiments, Cav-1 was also recovered when total tyrosine phosphoproteins were immunoprecipitated with antiphosphotyrosine (Fig. 1B). Finally, Cav-1 was recovered in anti-c-Src immunoprecipitates in an ATP-independent manner and was found to be phosphorylated on tyrosine-14 only in immune complexes recovered from ROS incubated with ATP (Fig. 1C).

Fig. 1 Cav-1 is phosphorylated in an ATP-dependent manner by kinase(s) in ROS. ROS were incubated in the presence or absence ATP, and subjected to immunoprecipitation with the indicated antibodies

Fig. 2 Cav-1 phosphorylation does not alter its localization to DRM fractions. ROS were incubated in the presence (right column) or absence (left column) of 1 mM ATP, DRM fractions were isolated, and subjected to immunoblot analysis with the indicated antibodies

To determine whether Cav-1 phosphorylation affects its partitioning into raft domains, we isolated DRM fractions from ROS incubated with or without ATP. DRM are low buoyant density, membrane fractions resistant to solubilization with some non-ionic detergents and are suggested to represent biochemically-isolated raft domains (Pike, 2004). As indicated in Fig. 2, although DRMs represent a small fraction of the total ROS protein (upper panels), Cav-1 is dramatically-enriched in these fractions (4th panels). Several tyrosine phosphoproteins (including Cav-1, 3rd panel, right column) are detectable in DRM fractions only after ATP incubation. As is apparent, phosphorylation of Cav-1 does not affect its partitioning into DRM fractions (4th panel).

Cav-1 is reportedly phosphorylated by several Src family tyrosine kinases (Labrecque et al., 2004; Li et al., 1996; Sanguinetti et al., 2003; Sanguinetti and Mastick, 2003). In order to investigate a putative role for Src family kinases in the in vitro phosphorylation of Cav-1 in ROS, purified ROS membranes were incubated with the Src selective inhibitor, PP2 (10 M) prior to their incubation with ATP. As shown, in Fig. 3, PP2 dramatically reduced the tyrosine phosphorylation of

Fig. 3 Cav-1 phosphorylation is reduced by the Src family kinase inhibitor PP2. ROS membranes were incubated with 10 M PP2 prior to addition of 1 mM ATP. Blots were probed with the indicated antibodies as described in the methods section. Cav-1 phosphorylation is dramatically reduced in PP2-treated ROS

Cav-1 (as well as several other tyrosine phosphoproteins) suggesting that the kinase responsible for Cav-1 phosphorylation in ROS, in vitro, is likely to be a Src family kinase.

4 Discussion

The results presented herein indicate that Cav-1 can be phosphorylated on tyrosine -14 in vitro by an endogenously-expressed Src family kinase in isolated ROS membranes. The conditions that trigger Cav-1 phosphorylation in vivo and the functional consequences resulting from this phosphorylation remain unknown. Light exposure stimulates tyrosine phosphorylation of several protein targets in ROS in vivo (Ghalayini et al., 1998) including the insulin receptor -subunit (Rajala et al., 2002) and triggers the binding of c-Src to ROS membranes, in vivo (Ghalayini et al., 2002). Our preliminary studies suggest that Cav-1 is not a target of light-dependent phosphorylation (Elliott and Ghalayini, unpublished results), but it remains unclear if damaging light or another form of stress could result in Cav-1 phosphorylation in vivo. In other cell-types, Cav-1 is phosphorylated in response to both oxidative and osmotic stresses (Cao et al., 2004; Chen et al., 2005; Sahasrabuddhe et al., 2006; Sanguinetti et al., 2003; Sanguinetti and Mastick, 2003; Volonte et al., 2001) and may regulate the apoptotic response (Shajahan et al., 2006). A possible role of Cav-1 phosphorylation in photoreceptor stress response remains to be elucidated.

Acknowledgments This work was supported by grants from the National Institutes of Health (EY012190, EY005235, EY11504, and RR017703), Research to Prevent Blindness, Inc., and the Foundation Fighting Blindness.

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Regulation of Neurotrophin Expression

and Activity in the Retina

Abigail S. Hackam

1 Introduction

Growth factors and their receptors are upregulated during retinal degeneration as an intrinsic tissue response that may protect remaining photoreceptors (Harada et al., 2002; Wen et al., 1995; Yu et al., 2004). Growth factor delivery to animal models of retinal degeneration leads to various degrees of preservation of photoreceptor morphology and function (Chaum, 2003). A major class of neuroprotective growth factors in the retina are the neurotrophins NGF, BDNF, NT3 and NT4/5. Therapeutic use of these molecules is complicated by their ability to activate pro-death as well as pro-survival pathways. Understanding mechanisms that induce neurotrophin upregulation during injury and controlling the cellular response to exogenous growth factors will be essential for taking full advantage of their neuroprotective activity while minimizing deleterious side-effects. This review summarizes the complex regulation of neurotrophin ligands and receptors that together promote neuronal survival.

2 TRK Receptor Activation: The Retinal Expression Paradox

Neurotrophins bind to two types of receptors, the high affinity Trk receptor tyrosine kinases (TrkA, B and C) and the low affinity p75NTR receptor. The neurotrophins have selective binding to the Trk receptors but bind equally well to p75NTR. Trk receptors contain a tyrosine kinase intracellular domain, a single-pass transmembrane domain and a large extracellular domain containing three leucine-rich motifs, two cysteine-rich regions and two immunoglobulin-like C2 type domains that bind neurotrophins.

A.S. Hackam

Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, 1638 NW10th Ave., Miami, FL 33136, Tel: 305-547-3723, Fax: 305-547-3658

e-mail: ahackam@med.miami.edu