Report |
Address correspondence to D.G. Drubin, Dept. of Molecular and Cell Biology, 16 Barker Hall, University of California, Berkeley, Berkeley, CA 94720-3202. Tel.: (510) 642-3692. Fax: (510) 643-0062. email: drubin{at}uclink4.berkeley.edu
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Abstract |
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Key Words: protein kinases; Eps15; Arp2/3; endocytosis; Saccharomyces cerevisiae
Abbreviations used in this paper: 1NA-PP1, 4-amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine; Ark, actin-regulating kinase.
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Introduction |
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Yeast actin-regulating kinase (Ark) 1p and Prk1p, a redundant pair of Ark family kinases, are strong candidates to directly couple the dynamic processes of actin cytoskeleton assembly and endocytosis (for review see Smythe and Ayscough, 2003). The three known in vivo targets of Prk1p, Pan1p (Eps15-related Arp2/3 activator; Zeng and Cai, 1999; Duncan et al., 2001), Ent1p (epsin-related protein; Watson et al., 2001), and Sla1p (an adaptor for Ste2p receptor endocytosis; Zeng and Cai, 1999; Howard et al., 2002), are actin patch proteins. Each of these Prk1p targets plays an important role in both endocytosis and in actin cytoskeleton regulation (for review see Engqvist-Goldstein and Drubin, 2003). Both elevation and loss of Ark kinase activity lead to defective actin cytoskeleton organization and endocytosis (Cope et al., 1999; Zeng and Cai, 1999; Watson et al., 2001; Zeng et al., 2001), suggesting that the regulation by protein phosphorylation is crucial for both systems. A mammalian Ark family kinase, AAK1, localizes to sites of clathrin-mediated endocytosis, and phosphorylation by AAK1 negatively regulates endocytosis (Conner and Schmid, 2002).
To gain insights into the regulatory mechanisms of actin cytoskeleton assembly and endocytosis by Prk1p, we used a chemical genetics approach (Bishop et al., 2001) that enabled us to rapidly modulate Prk1p activity in vivo. In comparison to the conventional approach using kinase-dead mutants, this approach enabled us to investigate the direct and immediate consequence of Prk1p inactivation for the regulation of actin assembly and endocytosis.
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Results and discussion |
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ark1 prk1-as1 and ark1
prk1-as3 cells showed specific sensitivity to 1NA-PP1. Upon treatment of these mutants with 1NA-PP1, unpolarized actin and actin clumps were observed in a dose-dependent manner (Fig. 1, A and B), indicating that inhibitor treatment mimics the phenotype seen upon loss of Ark1p and Prk1p (Cope et al., 1999). Actin cables appeared to be unaffected by inhibitor addition (Fig. 1 A; +1NA-PP1). As assessed by quantifying the percentage of cells forming actin clumps, the effect of 1NA-PP1 was saturated at 80 µM for ark1
prk1-as1, and at 40 µM for ark1
prk1-as3 (Fig. 1 B). As a further indication that ark1
prk1-as3 cells are more sensitive to the inhibitor, at optimal inhibitor doses, 40 µM for ark1
prk1-as3 and 80 µM for ark1
prk1-as1,
95% of ark1
prk1-as3 cells formed actin clumps, whereas
80% of ark1
prk1-as1 cells formed clumps (Fig. 1 B). The actin cytoskeleton of ark1
PRK1 cells was not affected by 40120 µM 1NA-PP1 (unpublished data).
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The Prk1p target Pan1p is an activator of the Arp2/3 complex. By introducing the temperature-sensitive arp2-1 mutant of an Arp2/3 complex subunit into the ark1 prk1-as3 background, we tested whether Arp2/3-mediated actin assembly is required for clump formation. The arp2-1 mutant shows significant endocytic defects at the permissive temperature, although it displays normal actin organization (Moreau et al., 1997). The ark1
prk1-as3 arp2-1 cells formed actin clumps much less efficiently (
5%, n = 100) than ark1
prk1-as3 cells (
95100%, n = 100) at 25°C (Fig. 1 G). The actin clumps disappeared rapidly in response to the actin monomer sequestering drug latrunculin-A (Ayscough et al., 1997; unpublished data), suggesting that actin turnover is not severely affected by Prk1p inhibition. In total, these data support the possibility that actin clumps are the result of unregulated Arp2/3-stimulated actin assembly.
Real-time analysis of actin patch dynamics upon Prk1p inhibition
To investigate actin patch dynamics as a function of Prk1p inhibition, we performed real-time analyses of ark1 prk1-as1 cells expressing Abp1-GFP (Fig. 2). Similar results were obtained with ark1
prk1-as3 cells (unpublished data). Abp1p is a component of cortical actin patches (Drubin et al., 1988). Within 1 to 2 min of 1NA-PP1 addition to ark1
prk1-as1 cells, Abp1-GFP patches aggregated into clumps (Fig. 2 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200305077/DC1). Once formed in the daughter cell, the actin clumps invariably moved toward the bud neck, and then into the mother cell (Fig. 2 B), consistent with the observation for fixed cells (Fig. 1 F), which exhibited an increase of mother clumps and a decrease of daughter clumps during the time course. The mechanistic basis for this movement is unknown, but does not seem to involve microtubules because the process was not sensitive to the microtubule-depolymerizing drug nocodazole (unpublished data). Within 1 min of Prk1p reactivation by inhibitor washout, the clumps disassembled, and the normal polarized distribution of Abp1 patches was restored (Fig. 2 C and Video 2). Rhodamine-phalloidin staining of fixed ark1
prk1-as1 cells confirmed that F-actin undergoes reversible aggregation upon 1NA-PP1 addition (unpublished data).
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Receptor-mediated endocytic internalization is blocked upon Prk1p inhibition
Further, we tested whether Prk1p kinase activity is required for -factor internalization by its receptor, Ste2p. Because of its high sensitivity to 1NA-PP1, all subsequent analyses used the prk1-as3 allele. Without inhibitor, ark1
prk1-as3 cells show internalization kinetics indistinguishable from ark1
PRK1 (Fig. 3 A). Treatment of ark1
prk1-as3 cells with 1NA-PP1 for 30 min specifically inhibited receptor internalization (Fig. 3 B). Even 120 µM inhibitor did not affect
-factor internalization by ark1
PRK1 cells (Fig. 3 B). Additionally, a kinase-dead mutant (ark1
prk1D159A) also showed a severe block of receptor internalization (Fig. 3 C). Thus, the inhibition of Prk1p kinase activity profoundly blocks the internalization step of receptor-mediated endocytosis.
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In this work, we examined the rapid and acute effects of Prk1p kinase inhibition and reactivation by applying a chemical genetics approach. We showed that abnormal actin clumps formed and disappeared within 1 to 2 min of Prk1p inhibition and reactivation, respectively. Further, we showed that the actin clumps contain endocytic proteins and 100-nm vesicles. We propose that Prk1p directly regulates the coupling between actin assembly and endocytosis by promoting disassembly and/or inactivation of an early endocytic complex that stimulates actin assembly (Fig. 5 A). When Prk1p is inhibited, this complex is stabilized, and actin assembly continues to be stimulated by endocytic proteins such as the Prk1p target Pan1p and the associated Arp2/3 complex (Zeng and Cai, 1999; Duncan et al., 2001), and/or other targets including Sla1p and Ent1p (Watson et al., 2001; Zeng et al., 2001; Fig. 5 B). In mammalian cells, the µ2 subunit of AP2 is phosphorylated by AAK1 at Thr-156 (ITSQVT156G) (Ricotta et al., 2002). However, the budding yeast AP2 homologue, Apm4p (Huang et al., 1999), is not important for receptor internalization and does not contain potential Prk1p-phosphorylation motifs similar to (L/IxxQxTG). Rather, our genetic experiment showing that Arp2p is required for clump formation supports the idea that Arp2/3-mediated actin assembly is negatively regulated by Prk1p, potentially via phosphorylation of the Arp2/3 activator, Pan1p. Our data also support the proposal that actin participates directly in yeast endocytosis.
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Materials and methods |
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Strains and plasmids
The yeast strains are listed in Table I. prk1::PRK1::URA3 and prk1
::prk1::URA3 integration plasmids were created as follows: First, a NotI site was introduced 249 bp upstream of the PRK1 ORF. The mutated KpnI/SacI PRK1 fragment was cloned into pBlueScript® II SK, and the PRK1 fragment was marked with URA3 at NotI to create a plasmid pDD877. prk1-as1 (M108G), prk1-as3 (M108G, C175A), and prk1D159A mutations were created in pDD877. The URA3-marked, PRK1-containing fragments were excised from the integration plasmids, and replaced the prk1
::LEU2 locus. These strains were each crossed with an ark1
strain, and the diploids were sporulated to obtain ark1
prk1 mutants. Analogue-sensitive Prk1 mutant proteins were expressed at normal levels, but Prk1D159Ap was expressed at only 2030% of normal levels at 25°C. The gene deletions were created as described previously (Cope et al., 1999). Functional GFP and CFP tags were integrated at the COOH terminus of Abp1p and Sla1p as described previously (Warren et al., 2002). To create Sla2-YFP, five alanines were introduced at the junction between the SLA2 ORF and YFP using pDH5 (from the Yeast Resource Center, Seattle, WA). The Sla2-YFP strain has growth properties that are indistinguishable from the wild type. pDD890 expresses Abp1-GFP from pRB2139 (Doyle and Botstein, 1996) on pRS317.
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Electron microscopy
Morphological observations of cells by conventional EM was performed essentially as described previously (Rieder et al., 1996), except that after fixation with FeCNOsO4/thiocarbohydrazide/FeCNOsO4 (OTO), the cells were further treated with 1% tannic acid for 30 min. Morphological observations of cells fixed by high pressure freezing and freeze substitution were performed as described in McDonald and Müller-Reichert (2002). Immuno-EM was performed as described previously (Rieder et al., 1996). Primary antibodies used were affinity-purified rabbit anti-actin antibody (a gift from A. Bretscher, Cornell University, Ithaca, NY) and mouse anti-GFP antibody (StressGen Biotechnologies); secondary 5- and 10-nm gold conjugates were obtained from Jackson Immunoresearch Laboratories.
Protein and immunological techniques
To obtain yeast whole-cell lysates, the cells were lysed with glass beads in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail, 50 mM NaF, 0.2 mM sodium orthovanadate, 25 mM ß-glycerophosphate, 1 µM cyclosporin A, 4and 0 µM cantharidin). 50 µg of the lysate was loaded per lane. For the time-course experiment, 2-OD cells were harvested and resuspended in 50 µl lysis buffer (50 mM Tris, pH 6.8, 2% SDS, 100 mM DTT, 8% glycerol, 0.02% BPB, protease inhibitor cocktail, 50 mM NaF, 0.2 mM sodium orthovanadate, 25 mM ß-glycerophosphate, and 1 µM cyclosporin A). The suspension was boiled for 3 min, and then lysed with glass beads for 2 min, followed by 1 min of boiling. 100 µl of the SDS-PAGE buffer was finally added to the lysate. 10 µl of the final supernatant was loaded per lane. Anti-Ent1p antibody (Watson et al., 2001) was used at 1:10,000 dilution for Western blotting.
-factor uptake assay
35S-labeled -factor was prepared as described in Howard et al. (2002). The
-factor uptake assay was performed at 25°C based on a continuous incubation protocol (Dulic et al., 1991) with modifications as follows: cells were grown in SD, harvested by centrifugation, and resuspended in internalization media (SD media with 0.5% casamino acid and 1% BSA). Then the cells were mixed with an equal volume of SD media containing 2x concentration of 1NA-PP1. After incubation with 1NA-PP1 for 30 min, 30,000 cpm/100 µl 35S-labeled
-factor was added at time zero. At the indicated time points, aliquots were withdrawn and diluted in ice-cold buffer at pH 6.0 (total
-factor) or pH 1.1 (internalized
-factor). The samples were then filtered, and radioactivity was measured in a scintillation counter. The results were expressed as the ratio of pH 1.1 cpm/pH 6.0 cpm for each time point to represent the percentage of internalization.
Online supplemental material
Video 1 and Video 2 show ark1 prk1-as1 cells expressing Abp1-GFP. The representative frames of the movies are shown in Fig. 2 A and Fig. 2 C. Fig. S1 shows EM of ark1
prk1-as3 cells post-treated with tannic acid. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200305077/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grants to D.G. Drubin (GM42759 and GM50399) and to K.M. Shokat (AI44009); an NIH grant (GM60979) and a Burroughs Wellcome Fund New Investigator Award to B. Wendland; and a National Science Foundation grant (DBI 0099705) to B. Wendland and J.M. McCaffery.
Submitted: 15 May 2003
Accepted: 21 July 2003
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