Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

490 H. ZHANG ET AL.

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Cells of bovine and rat retina contain high affinity receptors for insulin (Havrankova et al., 1978; Waldbillig et al., 1987; Reiter and Gardner, 2003; Yu et al., 2004). However, little research has been done on these receptors since these early reports probably due the absence of an identified intracellular target. We have demonstrated that light stimulates tyrosine phosphorylation of the b-subunit of insulin receptor (IRb) in vivo, which leads to the direct association of phosphoinositide 3-kinase (PI3K), an anti-apoptotic enzyme activity with the IRb (Rajala et al., 2002). In this communication, we examine the localization of the insulin receptor and PI3K in DRMs prepared from bovine ROS.

2. METHODS

2.1. Preparation of Bovine ROS and Fractionation of DRMs from ROS Incubated with and without Methyl-b-Cyclodextrin (MCD)

Bovine ROS were prepared from retinas on continuous sucrose gradients (25-50%) using a modification of the method of Zimmerman and Godchaux (Godchaux, III and Zimmerman, 1979; Zimmerman and Godchaux, III, 1982) as previously described (Bell et al., 1999). DRMs were prepared according to a previously described modification (Elliott et al., 2003) of the method of Seno et al. (Seno et al., 2001). For experiments involving cholesterol depletion, ROS, in buffer B, were incubated in the presence or absence of 15 mM MCD for 1 h at 37°C prior to DRM fractionation as previously described (Elliott et al., 2003).

2.2. Tyrosine Phosphorylation of ROS and DRM

Tyrosine-phosphorylated ROS and DRMs were prepared by incubation of each in a phosphorylation buffer [50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 2 mM MgCl2, 1.5 mM ATP] in the presence and absence of 0.2 mM Na3VO3 for 15 minutes at 37°C as originally described for ROS (Rajala and Anderson, 2001). After incubation, ROS and DRM fractions were subjected to Western blot analysis.

2.3. SDS-PAGE and Western Blot Analysis

Proteins were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The blots were incubated with anti-IRb (1 : 250), anti-PY (1 : 1000), or anti-p85 (1 : 4000) antibodies overnight at 4°C and developed by ECL according to the manufacturer’s instructions.

3. RESULTS

3.1. Localization of IRb and p85 to DRMs Isolated from Bovine ROS

Low buoyant-density DRMs were isolated from Triton-solubilized bovine ROS by discontinuous sucrose density gradient centrifugation and fractions collected from the top of the gradients were subjected to SDS-PAGE and immunoblot analysis. Protein determinations of these fractions indicate the presence of two protein peaks, the minor DRM peak in

Figure 68.1. Effect of MCD on the localization of caveolin-1 and other ROS proteins in low-buoyant-density DRMs. ROS incubated in the absence (-MCD) or presence (+MCD) of 15 mM MCD were solubilized with 1% Triton X-100 and separated into low and high-density fractions by discontinuous sucrose density gradient centrifugation. Fractions collected from the top of each gradient (top to bottom shown from left to right) were subjected to either protein determination (A) or Western blot analysis (B) with antibodies against IRb, p85 subunit of PI3K, and caveolin-1.

fraction 3 and the major peak in fraction 9 containing the majority of ROS proteins (Figure 68.1A, solid line). DRM fractions 2-5 were dramatically enriched in caveolin-1 with a peak in fraction 3 as previously observed (Nair et al., 2002; Elliott et al., 2003). In an attempt to determine whether DRMs isolated from ROS contained IRb and p85, immunoblot analysis of DRM fractions was performed. The results indicate the presence of a significant pool of IRb and p85 in the caveolin-enriched fraction 3 (Figure 68.1B). Fraction 9 was also immunoreactive for the presence of IRb and p85 and this fraction contained the majority of ROS proteins (Figure 68.1A, solid line). These results indicate that portions of both IRb and p85 are localized to photoreceptor DRMs.

To further confirm that the fractions isolated are authentic DRMs, ROS membranes were incubated with the cholesterol-sequestering agent, MCD, a treatment that disrupts cholesterol-rich DRMs. MCD treatment resulted in a dramatic loss of the low-buoyant- density band accompanied by a shift in the distribution of proteins to higher density sucrose fraction (Figure 68.1A and B). The concomitant shift in IRb, p85 and caveolin-1 to higher density fractions following MCD treatment shows that their colocalization to DRM is disrupted by the cholesterol-depleting actions of MCD.

3.2. Absence of Phosphatase Activity in DRM Fractions

Bovine ROS prepared as described in the Methods have an endogenous tyrosine kinase activity (Bell et al., 1999). ROS and DRM fractions were subjected to in vitro phosphorylation resulting in the tyrosine phosphorylation of several ROS proteins (Figure 68.2). Omission of sodium vanadate resulted in the absence of tyrosine phosphorylation of ROS proteins, suggesting the inhibition of phosphatase activity in ROS by sodium vanadate. An enrichment of a tyrosine phosphorylated 80 kDa protein was observed in the DRM fraction. It is interesting to note that omission of sodium vanadate in the DRM fraction did not affect

Figure 68.2. In vitro phosphorylation of ROS and DRM proteins. Phosphorylated ROS and DRM proteins (presence and absence of sodium vanadate) were subjected to SDS-PAGE followed by Western blot analysis with anti-PY99 antibody.

the tyrosine phosphorylation of the 80 kDa protein (Figure 68.2), suggesting the lack of phosphatase activity in DRM fractions.

4. DISCUSSION

Localization of insulin receptor in caveolae of adipocyte plasma membrane has been reported and cholesterol depletion attenuates insulin receptor signaling (Gustavsson et al., 1999). Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of insulin receptor (Gustavsson et al., 1999). These results suggest that functional insulin receptor resides in caveolae and requires the caveolar environment for signaling. In addition, tyrosine phosphorylation of caveolin-1 was catalyzed by insulin receptor (Kimura et al., 2002). Further, caveolin-1 deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue (Cohen et al., 2003). Baumann et al. (Baumann et al., 2000) reported that the resident lipid raft protein flotillin-1 recruits a complex of tyrosinephosphorylated Cbl and Cbl-associated protein to rafts, and that this recruitment is required for GLUT-4 translocation in response to insulin. In addition, epidermal growth factor stimulation results in the localization of PI3K, Akt2, and PTEN to lipid rafts of intestinal cells, and this localization is important for sodium absorption and enterocyte differentiation (Li et al., 2004). Furthermore, Vero cells (Monkey, African Green kidney) stimulated with lysophosphatidic acid results in the localization of PI3K to lipid rafts (Peres et al., 2003).

We have demonstrated that light stimulates tyrosine phosphorylation of IRb in vivo, which leads to the association of PI3K, an anti-apoptotic enzyme activity with the IRb (Rajala et al., 2002). We hypothesize that an important mechanism of retinal neuroprotection is through light activation of the insulin receptor, which stimulates the anti-apoptotic PI3K/Akt pathway. The molecular mechanism behind the light-activation of IRb is not known. In the present study, we have demonstrated the localization of IRb and p85 subunit of PI3K to DRM fractions of bovine ROS. Perhaps light-induced IRb activation of the antiapoptotic PI3K/Akt pathway occurs within lipid rafts of ROS, consistent with a previous study indicating that cholesterol-rich lipid rafts mediate Akt-regulated survival in prostate cancer cells (Zhuang et al., 2002).

The insulin signaling pathway is activated by tyrosine phosphorylation of the insulin receptor and key post-receptor substrate proteins, and balanced by the action of specific protein-tyrosine phosphatases (PTPase). Inhibition of PTPase activity results in enhanced tyrosine phosphorylation of the insulin receptor (Mahadev et al., 2001). The most compelling evidence for a physiological role of phosphatase PTP1B in insulin action has been the recent demonstration of enhanced insulin sensitivity and potentiation on insulinstimulated protein-tyrosine phosphorylation in PTP1B knockout mice (Klaman et al., 2000; Elchebly et al., 1999). Absence of phosphatase activity in DRM fractions could also stimulate insulin receptor phosphorylation. Studies are underway in our laboratory to test this hypothesis.

The phosphorylation we observed in DRMs (Figure 68.2) could be due to either receptor or non-receptor tyrosine kinases. It has been shown that the non-receptor tyrosine kinase Src phosphorylates insulin receptor on autophosphorylation sites (Yu et al., 1985; Peterson et al., 1996). Furthermore, activation of Src in rod photoreceptors cells has previously been reported (Ghalayini et al., 2002) and Src family kinases are present in detergent resistant cytoskeletal fractions (Ghalayini et al., 2002) and in DRMs isolated from bovine ROS (Martin et al., 2005). Earlier studies also demonstrated the in vivo tyrosine phosphorylation of caveolin by Src (Kimura et al., 2002). It is tempting to speculate that light-induced tyrosine phosphorylation of insulin receptor could be triggered in rafts by non-receptor tyrosine kinases, leading to the association with PI3K enzyme activity.

The phosphoinositide PI(4,5)P2, the preferred substrate for PI3K, accumulates in membrane rafts and promotes local co-recruitment and activation of specific signaling components at the cell membrane (Caroni, 2001). Raft-localized PI(4,5)P2 is regulated by lipid kinases (PI 5-Kinase) and phosphatases (eg. Synaptojanin) (Caroni, 2001; Chung et al., 1997). Localization of PI3K to the DRM fraction of ROS may have some important role in modulating PI(4,5)P2 levels in lipid rafts, since the absence of protein phosphatase activity could be compensating for the presence of PI3K in the DRM fractions. Further studies, however, are required to examine whether light triggers the phosphorylation of IRb or activation of PI3K activity in the DRM fractions.

5. ACKNOWLEDGEMENTS

This work was supported by grants from the National Institutes of Health (EY00871, EY04149, EY12190, EY15299 and RR17703); Research to Prevent Blindness, Inc. Raju V.S. Rajala is a recipient of Career Development Award from Research to Prevent Blindness, Inc.

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2. ROLE OF MERTK ACTIVATION IN RPE PHAGOCYTOSIS

Activity of the Mer tyrosine kinase receptor MerTK is essential for efficient engulfment of POS by RPE in vivo and in vitro (Mullen and LaVail, 1976; Edwards and Szamier, 1977) Despite its importance, mechanisms of MerTK activation and MerTK downstream signaling target proteins in RPE are still largely obscure. Retinal ligands of MerTK have not yet been conclusively identified. Moreover, we still do not know which RPE proteins serve as substrates for MerTK’s kinase activity during RPE phagocytosis. However, both endogenous and overexpressed MerTK reveal a striking redistribution to the sites of internalized POS in in vitro phagocytosis assays suggesting that MerTK receptors may be components of the phagocytic machinery of the RPE (Feng et al., 2002; Finnemann, 2003). Furthermore, challenge with isolated POS causes increased phosphorylation at tyrosine residues of MerTK in RPE in culture (Feng et al., 2002; Finnemann, 2003). Although their mutual dependence has not been demonstrated directly, levels of MerTK tyrosine phosphorylation commonly serve to assess the extent of MerTK activity.

3. FOCAL ADHESION KINASE SIGNALING ACTIVATES MERTK DURING RPE PHAGOCYTOSIS

Our previous studies on phagocytic signaling in RPE suggest an important role for focal adhesion kinase (FAK) in MerTK activation. FAK is a cytoplasmic non-receptor tyrosine kinase that colocalizes with integrin receptors at focal contacts where it commonly transduces signaling pathways downstream of activated integrins (for a recent review on FAK see (Parsons, 2003). Reversible activation of FAK is critical for integrin functions that involve cytoskeletal reorganization (Ilic et al., 1995). We expressed a C-terminal fragment of FAK that competes with full-length endogenous FAK for cytoskeletal anchorage. This fragment has been shown to act as a dominant-negative inhibitor of endogenous FAK abrogating FAK downstream signal transduction. We showed that the rat derived RPE-J cell line, like primary wild-type rat RPE, utilizes endogenous MerTK to engulf POS. Importantly, expression of the dominant-negative FAK C-terminal fragment in RPE-J cells inhibited POS engulfment (Finnemann, 2003). Furthermore, it eliminated the increase in MerTK tyrosine phosphorylation that is elicited by phagocytic challenge of RPE cells in culture (Finnemann, 2003). On the contrary, RPE cultures derived from RCS rats retained similar FAK activation as wild-type Long Evans rat RPE cultures in response to OS challenge. These results identify a novel signal transduction pathway in which FAK acts upstream of MerTK to stimulate the internalization machinery of the RPE.

4. avb5 SIGNALING VIA FOCAL ADHESION KINASE ACTIVATES MERTK

DURING RPE PHAGOCYTOSIS IN VIVO AND IN VITRO

Previous studies have shown that phosphorylation of FAK at tyrosine residue 861 promotes direct binding of FAK to the cytoplasmic face of avb5 integrin receptors (Eliceiri et al., 2002). In our studies, we observed increased levels of FAK in avb5 protein complexes isolated by immunoprecipitation from RPE-J cells during the early POS binding phase but loss of FAK from the integrin complex during the later POS internalization phase