QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online July 13, 2004
doi: 10.1242/10.1242/jcs.01219

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Park, H.-Y. Articles by Gilchrest, B. A. Search for Related Content PubMed PubMed Citation Articles by Park, H.-Y. Articles by Gilchrest, B. A.

The receptor for activated C-kinase-I (RACK-I) anchors activated PKC-ß on melanosomes

Department of Dermatology, Boston University School of Medicine, 609 Albany Street, Boston, MA 02118, USA

View larger version (25K): [in a new window]   Fig. 1. Co-localization of RACK-I and tyrosinase. (A) Subconfluent culture of melanocytes plated on Labtek Chamber slides was first incubated with the monoclonal antibody against RACK-I, then additionally incubated using polyclonal antibody against tyrosinase. Subsequently, the cells were further incubated with rhodamine-tagged secondary antibody against RACK-I antibody (A) and FITC-tagged secondary antibody and against tyrosinase antibody (B) and examined by confocal microscopy. When the images were overlayed, the computer-assigned yellow-orange color indicates co-localization of the two proteins (C).

View larger version (30K): [in a new window]   Fig. 2. Distribution of RACK-I within melanocytes. (A) Purified melanosomes from subconfluent melanocyte cultures were subjected to immunoblot analysis using a monoclonal antibody against RACK-I. As a positive control, fibroblasts over expressing RACK-I were used, as suggested by the vendor. (B) Melanosomes were separated from the cytoplasmic and particulate fractions of melanocytes as previously described (Seiji et al., 1963). Proteins from cytoplasmic, melanosome and particulate fractions were subjected to immunoblot analysis using a monoclonal antibody specific for RACK-I. As the loading control, the membrane was stained with Coomassie Blue.

View larger version (26K): [in a new window]   Fig. 3. Depletion of PKC reduced the level of RACK-I associated with melanosomes. Paired cultures of subconfluent melanocytes were treated with vehicle alone or 10–7 M TPA for 2 weeks, the time period known to delete PKC in pigment cells (Park et al., 1996; Park and Gilchrest, 1999). Cells were harvested and the total level of RACK-I in vehicle- and TPA-treated samples was determined. Then cytoplasmic (B) and melanosome (C) fractions were separated and immunoblotted for RACK-I. Membranes were stained with Coomassie Blue as loading control.

View larger version (59K): [in a new window]   Fig. 4. Co-localization of activated PKC-ß and RACK-I. To determine whether activated PKC-ß co-localizes with RACK-I, paired melanocyte cultures plated onto Labtek Chamber slides were treated with phorbol dibutyrate (PDBu) for 90 minutes to activate PKC. Control wells received DMSO only (A1 and B1). Then they were subjected to double immunostaining using polyclonal antibody against PKC-ß (A2) or PKC- (B2) and monoclonal antibody against RACK-I (A3 and B3). Then cells were further incubated with FITC-conjugated secondary antibody against PKC-ß or PKC- antibodies and rhodamine-conjugated secondary antibody against RACK-I antibody and analyzed as in Fig. 1. The images were overlaid to determine co-localization (indicated in yellow) between PKC-ß and RACK-I (A4) and PKC- and RACK-I (B4).

View larger version (30K): [in a new window]   Fig. 5. RACK-I and PKC-ß are complexed on melanosomes. (A) Purified melanosomes were subjected to immunoprecipitation using a monoclonal antibody against RACK-I. Immunoprecipitated proteins were then separated in 7.5% SDS-PAGE, transferred onto a nitrocellulose membrane, and the membrane was then cut at the molecular weight of 50 kD. The upper part of the membrane was immuno-reacted using a monoclonal antibody against PKC-ß and the bottom part of the membrane was immuno-reacted using a monoclonal antibody specific for RACK-I. (B) Purified melanosomes were subjected to immunoprecipitation using a monoclonal antibody against RACK-I. Then the immunoprecipitated proteins were separated and immunoblot analysis was performed using specific antibody against PKC- on the upper part of the membrane and monoclonal antibody against RACK-I. Purified recombinant PKC- (5 ng) purchased from Panvera was used as a control.

View larger version (40K): [in a new window]   Fig. 6. TPA increases the amount of activated PKC-ß associated with RACK-I. Subconfluent melanocyte cultures were treated with 10–7 M TPA or vehicle alone for 90 minutes. Equal amounts of proteins from vehicle- and TPA-treated cell lysates were subjected to immunoprecipitation using an antibody against RACK-I. The immunoprecipitated proteins were separated and subjected to immunoblot analysis as described for Fig. 5. Densitometric analysis of each band was performed. When the ratio of PKC-ß to RACK-I in vehicle-treated samples was set arbitrarily at 1.0, the ratio in TPA-treated samples was 2.67.

View larger version (19K): [in a new window]   Fig. 7. DECA disrupts the interaction between PKC-ß and RACK-I. (A) Subconfluent melanocyte cultures were treated with DECA or vehicle alone for 30 minutes. Then the cultures were UV irradiated (UVB, 12 mJ/cm2) to activate DECA and treated with 10–7 M TPA for an additional 90 minutes. Then cells were harvested and equal amounts of proteins from DECA- or vehicle-treated cells were subjected to immuno-precipitation using an antibody against RACK-I, followed by immunoblot analysis using antibodies against PKC-ß and RACK-I, as described for Fig. 5. Densitometric analysis of each band was performed. When the ratio of PKC-ß to RACK-I was set arbitrarily at 1.0, the ratio in DECA-treated samples was 0.25. (B) Subconfluent melanocyte cultures were treated with DECA or vehicle alone, UV-irradiated to activate DECA and then treated with 10–7 M TPA as described above. Cells were harvested and tyrosinase activity was determined as previously described (Pomerantz, 1964). Statistical analysis using a paired Student's t-test was performed.

View larger version (19K): [in a new window]   Fig. 8. RACK siRNA decreases tyrosinase activity. (A) A paired culture of melanocytes were transfected with 10nM control RNA or RACK-I siRNA. 72 hours later, cells were harvested and immunoblot analysis was performed for RACK-I, tyrosinase and PKC-ß. (B) In parallel, tyrosinase activity was measured. Statistical analysis was performed using a paired Student t-test.

View larger version (13K): [in a new window]   Fig. 9. RACK-I siRNA blocks the TPA-induced increase in tyrosinase activity. Paired melanocyte cultures were transfected with 10 nM control RNA or siRNA and 72 hours after transfection cells were treated with 10–7 M TPA or vehicle alone for 90 minutes. Then cells were harvested and tyrosinase activity was measured. Statistical analysis using a paired Student's t-test was performed.

View larger version (21K): [in a new window]   Fig. 10. Proposed mechanism by which RACK-I anchors activated PKC-ß on melanosome membranes. In human melanocytes, when PKC is activated by TPA treatment, or a physiologic signal, activated PKC-ß (PKC-ß*), but not activated PKC- (PKC-*), binds RACK-I and translocates to the melanosome membrane. Activated PKC-ß, anchored on the melanosome via RACK-I, then phosphorylates inactive tyrosinase (TYRi) at serine residues of amino acid positions at 505 and 509 of its cytoplasmic domain and activates tyrosinase (TYRa), leading to melanogenesis.