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Targeting of Protein Kinase C to Caveolae 603 Figure 2 Targeting of Protein Kinase Ca to Caveolae Chieko Mineo,* Yun-Shu Ying,* Christine Chapline,‡ Susan Jaken,‡ and Richard G.W. Anderson* *Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039; and ‡Adirondack Biomedical Research Institute, Lake Placid, New York 12946 Abstract. Previously, we showed caveolae contain a latory domain of the molecule. A 68-kD PKCa-binding population of protein kinase Ca (PKCa) that appears protein identified as sdr (serum deprivation response) to regulate membrane invagination. We now report was isolated by interaction cloning and localized to ca- that multiple PKC isoenzymes are enriched in caveolae veolae. Antibodies against sdr inhibited PKCa binding. of unstimulated fibroblasts. To understand the mecha- A 100–amino acid sequence from the middle of sdr Downloaded from http://rupress.org/jcb/article-pdf/141/3/601/1257697/29449.pdf by guest on 27 September 2021 nism of PKC targeting, we prepared caveolae lacking competitively blocked PKCa binding while flanking se- PKCa and measured the interaction of recombinant quences were inactive. Caveolae appear to be a mem- PKCa with these membranes. PKCa bound with high brane site where PKC enzymes are organized to carry affinity and specificity to caveolae membranes. Binding out essential regulatory functions as well as to modu- was calcium dependent, did not require the addition of late signal transduction at the cell surface. factors that activate the enzyme, and involved the regu- he protein kinase C (PKC)1 family of phospholipid- stimulus. A variety of PKC-binding proteins (10) and lip- dependent kinases are important regulators of ids (22) have been identified that might function to com- Tgrowth, differentiation, and gene expression (8, 22). partmentalize PKC isoenzymes. Based on the requirements for activation, the 12 mamma- One place on the plasma membrane where PKCa ap- lian PKC isoenzymes can be grouped into three categories pears to be a resident protein is caveolae (24, 25). Both (10): PKCa, bI, bII, and g require calcium, phosphati- cell fractionation and immunogold labeling of whole dylserine (PS), and diacylglycerol (DAG) for activity; plasma membranes show that PKCa is highly concen- PKCe, d, h, s, and m require PS and DAG; and PKCj, i, trated in caveolae of unstimulated cells (25). Despite the and l need only PS. All isoenzymes have similar catalytic presence of many different resident and migratory pro- domains but differ in the structure of their regulatory do- teins in this domain (14), a 90-kD protein is the major mains. The intramolecular interaction between a 17– PKCa substrate detected in intact cells as well as isolated amino acid–long “pseudosubstrate” and the catalytic site caveolae (25). Phosphorylation in vitro occurs in the ab- may be a critical step in controlling the activity of many of sence of activators such as DAG or PS (25), suggesting the these enzymes (5). enzyme is constitutively active when located in this com- Most cells express multiple isoforms of PKC, and each partment. The uptake of molecules by caveolae is linked has a specific set of functions (5). These isoenzymes, how- to PKCa kinase activity (25), so the enzyme may play a ever, display little substrate specificity in in vitro assays. key role in regulating the internalization of caveolae. Therefore, other mechanisms must govern the specific Therefore, a mechanism must exist for directing PKCa to function of each isoenzyme in the cell. One way to achieve caveolae and regulating substrate specificity at this site. specificity is by targeting individual isoenzymes to select We now report that caveolae isolated from Rat-1 cells dis- locations in the cell (18), using high-affinity interactions play a Ca11-dependent, high-affinity PKCa binding activ- between the enzyme and a subcellular compartment. The ity that may be involved in targeting the enzyme to this isoenzyme could be constitutively present in the target domain. Using interaction cloning together with immu- compartment or recruited there after the cell receives a nolocalization and a competitive binding assay, we have identified a protein component of this binding site as se- rum deprivation response protein (Sdr) (7). Address all correspondence to Richard G.W. Anderson, Department of Cell Biology and Neuroscience, University of Texas Southwestern Medi- cal Center, Dallas, TX 75235-9039. Tel.: (214) 648-2346. Fax: (214) 648- Materials and Methods 7577. E-mail: [email protected] 1. Abbreviations used in this paper: DAG, diacylglycerol; MBP, maltose- Materials binding protein; PKC, protein kinase C; PMA, phorbol-12-myristate-13-ace- tate; PS, phosphatidylserine; sdr, serum deprivation response; RD, regula- Fetal calf serum was from Hazleton Research Products, Inc. (Lenexa, tory domain. KS). DME, trypsin-EDTA, penicillin/streptomycin, and OptiPrep were The Rockefeller University Press, 0021-9525/98/05/601/10 $2.00 The Journal of Cell Biology, Volume 141, Number 3, May 4, 1998 601–610 http://www.jcb.org 601 from GIBCO BRL (Gaithersburg, MD). Percoll was from Pharmacia Bio- room temperature, and washed three times with 250 ml of buffer D plus 1 tech (Piscataway, NJ). EGF was from CalBiochem (San Diego, CA). Hu- mg/ml heat-denatured BSA. The indicated PKC mixtures (100 ml) were man recombinant PKCa and PKCe were from PanVera Corporation added and incubated for 30 min at the indicated temperature. The wells (Madison, WI). 125I-radiolabeled streptavidin with specific activity of were washed rapidly seven times at 48C with 250 ml of buffer D plus 1 mg/ml 20–40 mCi/mg and ECL reagent were obtained from Amersham Corp. heat-denatured BSA. Each sample was fixed with 250 ml of 3% paraform- (Arlington, IL). Antibodies were obtained from the following sources: aldehyde in buffer D for 30 min at room temperature. The amount of anti–caveolin-1 mAb IgG, anti–caveolin-1 polyclonal antibody IgG, anti- PKCa bound to EDTA-stripped membrane was determined by radioim- PKCa, -PKCe, -PKCl IgGs (mAb), anti-RACK1 IgG (mAb), and anti– munoassay as previously described (28) using anti-PKCa (1 mg/ml), biotin- integrin b3 IgG (mAb) were from Transduction Laboratories (Lexington, ylated goat anti–mouse IgG (2 mg/ml), and 125I-streptavidin (2 mCi/ml). KY); peroxidase-conjugated anti–mouse IgG and anti–rabbit IgG were To measure binding in solution, 15 mg of freshly isolated caveolae or from Organon Teknika (West Chester, PA); biotinylated goat anti–mouse noncaveolae membrane fractions were incubated in a polyallomer centri- IgG was from Vector Laboratories (Burlingame, CA); and TRITC-goat fuge tube (Beckman Instruments) for 30 min in the presence of purified anti–mouse IgG [H1L] and FITC-goat anti–rabbit IgG [H1L] were from PKCa (5 nM) under the indicated conditions. After the incubation, the Zymed Laboratories Inc. (South San Francisco, CA). Polyclonal anti-sdr sample was chilled at 48C for 10 min and centrifuged for 60 min at 100,000 g peptides were produced by standard methods. The PKCa pseudosubstrate to separate the membrane (pellet). The pellet was rinsed gently with peptide (RFARKGALRQKNVHENKN) was synthesized by University buffer A, and 30 ml of Laemmli sample buffer was added. The sample was of Texas Southwestern Medical Center Polymer Core Facility. Immulon I heated at 958C for 3 min and loaded onto 12.5% SDS polyacrylamide gels. Removawell 96-well plates were purchased from Dynatech Laboratories PKCa was detected by immunoblotting using mAb anti-PKCa IgG. (Chantilly, VA). Immobilon transfer nylon was from Millipore (Bedford, Isolation of sdr. Interaction cloning (4) was used to isolate a 68-kD MA). Crystalline bovine serum albumin and phorbol-12-myristate-13-ace- PKCa-binding protein designated as clone 34. Analysis of the sequence tate (PMA) were from Sigma Chemical Co. (St. Louis, MO). 1,1,1-trichlo- showed that clone 34 was identical to a previously cloned protein known roethane was from Aldrich Chemical Co., Inc. (Milwaukee, WI). as sdr (7). Clone 34/sdr cDNA was ligated in frame into the pTrc (InVitro- gen, Carlsbad, CA) or pQE (Qiagen, Chatsworth, CA) bacterial expres- Downloaded from http://rupress.org/jcb/article-pdf/141/3/601/1257697/29449.pdf by guest on 27 September 2021 Methods sion vector to produce recombinant His-tagged fusion proteins. The ex- pressed sequences corresponding to polypeptides containing amino acids Cell Culture. Rat-1 cells (6 3 105) were seeded in 100-mm-diam dishes 1–168, 145–250, and 250–417 were purified by nickel-nitrilotriacetic acid and grown in 10 ml of DME supplemented with 10% (vol/vol) fetal calf chromatography according to the manufacturer’s instructions. The puri- serum for 4 d. Cells were then incubated for 24–48 h in DME without se- fied peptides were used to raise antisera in rabbits. Antisera were purified rum before each experiment. Normal human fibroblasts were cultured on by affinity chromatography using the expressed sequences coupled to coverslips as previously described (6). Sepharose. Isolation of Caveolae Fractions. Detergent-free caveolae fractions were ELISA Assay. Fragments of clone 34/sdr containing residues 1–168, prepared by the method of Smart et al. (26). All steps were carried out at 145–250, or 250–417 (2.8 mg/ml in PBS, 100 ml per well) were bound to in- 48C. Cells were collected by scraping in 5 ml of ice-cold buffer A (0.25 M dividual wells of a 96-well dish, and the wells were blocked with BSA (2% sucrose, 1 mM EDTA, 20 mM tricine, pH 7.8, with or without 1 mM in PBS). PKCa (20 ng of recombinant PKCa) or RDa (60 ng of recombi- CaCl2) and pelleting at 1,400 g for 5 min. After douncing, the postnuclear nant maltose-binding protein [MBP] fused to RDa) were added to the supernatant fraction was obtained, layered on top of 23 ml of 30% Percoll wells in buffer E (PBS plus 0.1 mg/ml BSA, 1 mM EGTA, 0.466 mM solution prepared in buffer A, and centrifuged at 84,000 g for 30 min CaCl2, and 2.1 mM MgCl2).
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