Regulation of Slik by activation segment phosphorylation

Protein kinases carry out important functions in cells both by phosphorylating substrates and by means of regulated noncatalytic activities. Such non-catalytic functions have been ascribed to many kinases, including some members of the Ste20 family. The Drosophila Ste20 kinase Slik phosphorylates and activates Moesin in developing epithelial tissues to promote epithelial tissue integrity. It also functions non-catalytically to promote epithelial cell proliferation and tissue growth. We carried out a structure-function analysis to determine how these two distinct activities of Slik are controlled. We find that the conserved C-terminal coiled-coil domain (CCD) of Slik, which is necessary and sufficient for apical localization of the kinase in epithelial cells, is not required for Moesin phosphorylation but is critical for the growth-promoting function of Slik. Slik is autoand trans-phosphorylated in vivo. Phosphorylation of at least two of three conserved sites in the activation segment is required for both efficient catalytic activity and non-catalytic signaling. Slik function is thus dependent upon proper localization of the kinase via the CCD and activation via activation segment phosphorylation, which enhances both phosphorylation of substrates like Moesin and engagement of effectors of its non-catalytic growth-promoting activity. Protein kinases play key roles in most cellular processes through their well-known catalytic function of reversibly phosphorylating their substrates. Catalytic activity-independent functions have been ascribed to an increasing number of these proteins, leading to the emerging view of protein kinases as molecular switches (analogous to small GTPases) rather than just enzymes (1). The Sterile-20 (Ste20) kinases are a large group of Ser/Thr kinases, with 28 members in humans divided into two distinct families (PAKand GCK-like) and ten subfamilies. Outside of the catalytic domain, which shows http://www.jbc.org/cgi/doi/10.1074/jbc.M115.645952 The latest version is at JBC Papers in Press. Published on July 13, 2015 as Manuscript M115.645952 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of Slik by activation segment phosphorylation 2 homology to the yeast kinase Ste20p, the ten subfamilies show little sequence similarity to one another. Despite their structural diversity, many Ste20 kinases appear to regulate a few common cellular functions including cell proliferation and survival, cytoskeletal dynamics, and ion transport (2). Some of the Ste20 kinases are well-known proteins with clearly defined physiological functions – e.g. the Pak subfamilies in cytoskeletal regulation (3), the GCK-II subfamily kinases Mst1/Mst2/Hippo in tissue growth and tumour suppression (4), and the GCK-VI subfamily kinases Osr1 and Spak in regulating ion channels (5). Many others are less well-characterized. While most of the identified functions of these kinases have been attributed to substrate phosphorylation, catalytic activity-independent functions have been proposed for some (6). The GCK-V subfamily is composed of two kinases in mammals, Slk and Lok/Stk10. These kinases are characterized by an N-terminal Ste20like kinase domain and a C-terminal coiled-coiled repeat-containing domain (CCD), connected by a non-conserved central linker domain (NCD) of variable length. SLK has been implicated in the regulation of a variety of cellular processes, including cell cycle progression (7), apoptosis (8,9), and cell migration (10-12). The one fundamental function of these kinases that is evolutionarily conserved is the regulation of Ezrin/Radixin/Moesin (ERM) family proteins. ERM proteins are important regulators of the cell cortex, acting as crosslinkers to connect the actin cytoskeleton to diverse transmembrane proteins at the plasma membrane. Their ability to do so requires phosphorylation of a highly conserved Thr residue near the C-terminus, which disrupts autoinhibitory interactions between the Nand Cterminal domains (13). In Drosophila, phosphorylation at the critical residue (and thus activation) of Moesin was negligible in epithelial cells mutant for Slik, the single GCK-V orthologue in flies (14). Recently, depletion of Slk and/or Lok in mammalian cells was shown to strongly reduce ERM protein phosphorylation (15,16). Mutating or depleting these GCK-V kinases in Drosophila or mammalian cells produces cellular and tissue phenotypes similar to those caused by mutating or depleting the ERM proteins themselves, including impaired epithelial tissue integrity (14,17), disrupted organization of apical microvillli (14,18), reduced cortical stiffness (19,20), and misorientation of the mitotic spindle and cytokinesis defects (15,19,20). Taken together, these studies strongly highlight the importance of GCK-V kinase function in ERM regulation to control cell structure and epithelial organization, and their potential involvement in pathological conditions where these are affected. Drosophila slik mutants have an additional developmental phenotype that is separable from Moesin regulation. The mutant animals grow slowly, requiring approximately three times as long to reach full size in the larval stage before subsequently dying (21). Overexpression of Slik in wing imaginal discs (the epithelial precursors to the adult wing) has the opposite effect, increasing cell proliferation rates and causing overgrowth of the wing (21). Thus, as with other Ste20 kinases, including Hippo/Mst and Tao1 (22-27), Slik regulates tissue growth. There are two unusual features of Slik-driven growth. First, Slik expression had nonautonomous effects, with not only Slik-expressing cells but also surrounding cells displaying the proliferative response (21). Second, a point mutant form of the kinase that is expected to impair catalytic activity also induced the proliferative response (21). This suggests that the effect does not require catalytic activity, in line with the catalytic activity-independent allosteric functions of a number of kinases and pseudokinases (1). Because of its involvement in both processes, Slik is well-positioned to serve as one of the mechanisms for coordinating epithelial cell organization with epithelial tissue growth (28). In order to understand how these distinct activities of Slik may be regulated, we undertook a structurefunction analysis of this kinase. Our results confirm that Slik kinase activity is not required for its ability to promote proliferation, and point to both apical localization via the CCD and phosphorylation as key mechanisms regulating both the epithelial integrity (catalytic) and growthpromoting (non-catalytic) functions of Slik. EXPERIMENTAL PROCEDURES Cloning and constructs To create the slik and moesin transgenes, PCR was used to introduce an EcoRI site immediately upstream of the initiator Met codon and a KpnI site either immediately downstream of the stop codon of fullby gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of Slik by activation segment phosphorylation 3 length slik and moesin cDNAs, or replacing the stop codon of moesin, by PCR. For Slik, a KpnI and a NarI site were then silently introduced at codon 195 and 200 of the coding sequence, respectively. For Slik, Slik and Slik mutants, the first ~700bp of the modified 5′ slik coding sequence were PCR amplified with specific primers to introduce the mutations and the EcoRI/KpnI-digested products encoding the N-terminus of Slik were ligated together with the KpnI fragment encoding the Cterminus in a modified pMT.puro3c expression vector that introduces an N-terminal Myc epitope tag, and into pUASTattB for generation of transgenic flies. For the Slik mutant, an EcoRI/NarI fragment was used instead because the KpnI site is destroyed by the introduced mutation. The kinase dead Slik mutant, in which Asp is mutated to Asn, has been described previously (21). The modified moesin cDNA was cloned into pMT.puro3c vectors modified to encode either an N-terminal Myc epitope tag or a C-terminal GFP tag using a EcoRI/KpnI digest. The Moesin and Moesin mutants were introduced by PCR site directed mutagenesis. The construct for expressing the GST-MoeCT fusion protein was generated by cloning a fragment encoding the Cterminus of Moesin (amino acids 311-575 of MoePD) in pET-23b. For the Slik domain constructs, sequences coding for amino acids 1-321 (Slik), 322-988 (Slik), or 989-1300 (Slik) of Slik were PCR amplified with primers that introduced a 5′ EcoRI site and 3′ KpnI site. EcoRI and KpnIdigested products were cloned into pMT.puro3c and pUAST plasmids modified to encode add an N-terminal Myc epitope tag. To generate the slik 5'-untranslated region (UTR) dsRNA transgene, the UTR sequence (spanning three exons between nucleotides 24401370 and 24402688 of the Drosophila chromosome 2R scaffold) was PCRamplified from S2 cell cDNA and cloned in inverted repeat orientation in pWIZ. This same sequence, with T7 and T3 promoters introduced at either end by PCR, was used as a template for generating slik 5'-UTR dsRNA for cell culture experiments. A plasmid encoding PLCδ-PH fused to GFP was generously provided by Dr. J. Brill, and was re-cloned into pMT.puro3c for expression in S2 cells. Fly strains and reagents Slik transgene constructs cloned in pUAST-attB plasmids were recombined into the 65B2 locus using the PhiC31 integrase system (29). UAS-slik-5'-UTR-dsRNA, UAS-myc-Slik, UAS-myc-Slik, and UASmyc-Slik transgenic flies were generated by standard P-element transgenesis. UAS-Ft flies were from Dr. H. McNeill. UAS-CyclinD,UASCdk4 and UAS-dMyc flies were from Dr. B. Edgar. UAS-Dp110 flies were from Dr. S. Leevers. UAS-Eiger flies were from Dr. M. Miura. UAS-Mer flies were from Dr. R. Fehon. UASmerRNAi flies (transformant ID 7161) were from the Vienna Drosophila Resource Centre (VDRC). All other fly stocks were from the Bloomington Drosophila Stock Centre. The following antibody dilutions were used: guinea pig anti-Slik (21) at 1:250-1:1000 for immunostainings and 1:20000 for immunoblotting; rabbit anti-PhosphoEzrin(Thr)/Radixin(Thr)/Moesin(Thr) (Cell Signaling Technology, #3141) at 1:100 for immunostainings and 1:2000 for immunoblotting; rabbit anti-cleaved Caspase-3 (Cell Signaling Technology, #9661); mouse anti-c-Myc (Santa Cruz) at 1:1000 for immunostaining and 1:2000 for immunoblotting; rabbit anti-GFP (Torrey Pines Biolabs Inc.) at 1:1000. Cell culture slik 5'-UTR dsRNA was synthesized in two separate in vitro transcription reactions using MEGAscript T3 and T7 kits (Ambion). RNA products were mixed in equal amounts, annealed at 95oC for 5 mins, and then slowly cooled to room temperature. All cell-based experiments were done in Drosophila S2-R+ cells grown in Schneider's medium (Lonza) supplemented with 10% FBS (Gibco) and 50 U/ml Pen/Strep (Gibco) at 25oC. Cells were plated at 1x106/ml. For RNA interference, the appropriate dsRNA was added to the culture medium on day 0 at 1 μg/ml and left to incubate for 48hrs. Plasmids were then transfected on day 2 using the XtremeGENE HP reagent (Roche) as described by the manufacturer. Typically, 500 ng/ml of Slik plasmids were added, and 200 ng/ml of Moesin plasmids. On day 3, an additional dose of dsRNA was added along with 0.5 mM CuSO4 for induction. The cells were finally processed on day 5. by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of Slik by activation segment phosphorylation 4 Immunoprecipitations, pulldowns, and Western blotting Cells were harvested in icecold PBS and then lysed for 15mins at 4oC in lysis buffer [50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40 with added 1x protease inhibitor (Roche), 1x AEBSF (Sigma) and 1x phosphatase inhibitor (Roche)]. Lysates were centrifuged to remove cell debris (12,000g, 15mins, 4oC). For immunoprecipitations, cleared lysates were incubated with the appropriate antibody for 1hr at 4oC and then with protein A/G agarose beads (Santa Cruz) for 1hr at 4oC. The beads were washed in lysis buffer 3 times for 5 mins and then suspended in 2x Laemmli sample buffer at 80oC for 5 mins to solubilize proteins. Proteins were separated using a standard SDS-PAGE protocol and then transferred to a PVDF membrane using a semi-dry transfer apparatus. Immunoblotting was conducted according to standard procedures. For pulldowns, cleared lysates from cells expressing either Slik or the PH domain of Phospholipase Cδ were incubated with agarose beads coupled to various PIPs according to manufacturer’s instructions (Echelon Biosciences) and bound proteins analyzed by Western blotting. In vitro kinase assays As a substrate for kinase assays, a fusion protein consisting of GST fused to amino acids 311-575 from the C-terminus of Moesin-PD was expressed in E. coli BL21 cells and purified using glutathione agarose (Pierce) according to manufacturer’s protocol. S2 cells were transfected with Slik or Slik expression vectors and induced as described above. Three days after induction, cells were washed with PBS and lysed for 15 min on ice in IP lysis buffer (25 mM HEPES, pH7.2, 100 mM NaCl, 10 % glycerol, 0.5% NP-40 containing 5 mM EGTA, 5mM EDTA, 50 mM NaF, 10 mM ßglycerophosphate, 1 mM NaVO4, 0.5 mg/ml AEBSF, and 1x PI cocktail). Samples were centrifuged at 14,000 rpm for 10 min at 4 ̊C. Rat anti-Slik antibody (21) was added to the soluble fractions, and samples were incubated for 2h at 4 ̊C. Protein A agarose beads were added and samples incubated an additional 2 h. The beads were washed three times at 4 ̊C in 25 mM HEPES, pH7.2, 150 mM NaCl, 0.5% NP-40 containing 5 mM EGTA, 5mM EDTA,10 mM ßglycerophosphate, 1 mM NaVO4, 1x PI cocktail, and 0.01% BSA. Beads were rinsed two times in kinase buffer (20 mM Tris, pH 7.5, 15 mM MgCl2, 3 mM MnCl2, 1mM DTT, 1mM NaVO4, 10 mM ß-glycerophosphate, 0.1 mg/ml AEBSF, 1x PI cocktail), and then suspended in kinase buffer containing 50 μM ATP, 5 μCi (γ-32P)-ATP (for radioactive labeling), and 5 μl of GSTMoe.CT substrate. Reactions were incubated for 20 min at 30 ̊C, stopped by addition of 5x sample buffer and immediate heating at 90 ̊C for 5 min, and separated by SDS-PAGE. Gels were incubated twice for 10 min in 20% methanol/10% glycerol, dried, and developed by autoradiography. Fly crosses, EdU labeling, and immunostainings Unless otherwise stated, crosses were performed at 25°C. For some of the drivers of growth and apoptosis, crosses were performed at 18°C. For expression of UAS-Slik transgenes in wild-type discs, Gal80 was used to inhibit transgene expression during embryonic stages. Parents were allowed to lay eggs at 21°C for two days. One day later, offspring were transferred to 27°C to inhibit GAL80 and activate GAL4-dependent transgene expression. For experiments involving rescue of dsRNA-mediated Slik depletion, crosses included a UAS-Dcr transgene and were carried out at 27°C to maximize the slik dsRNA phenotype while minimizing the effects of transgenic Slik expression. mer dsRNA crosses were performed at 27°C. For immunostainings, third instar larvae were dissected in PBS to isolate the anterior halves and remove the fat, the digestive system and salivary glands. The resulting carcasses were collected for 20 min in ice cold PBS and then fixed using 4% PFA in 0.2% PBS/0.2% Tween (PBT) for 20 mins. The anterior halves were then washed three times in 0.2% PBT followed by blocking for 1 hour in BBT (0.2% PBT + 0.3% BSA). The primary antibodies diluted in BBT were added and incubated overnight at 4°C. The next day, samples were washed 3 times in 0.2% PBT and then incubated for 2 hours with the fluorescent secondary antibodies diluted in BBT (Invitrogen, Jackson). Samples were again washed three times in 0.2% PBT and then incubated for 10 mins with 2.5 ug/ml DAPI. After a final wash in PBT, the larvae were transferred into mounting medium (90% glycerol, 10% PBS, 0.2% n-propyl gallate) and mounted. For EdU stainings, larvae by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of Slik by activation segment phosphorylation 5 were dissected in serum free medium (EX-CELL 420, Sigma) and cleaned anterior halves labeled for 1 h with EdU and processed using the Click-iT Edu Alexa Fluor 55 Imaging Kit (Life Technologies) according to manufacturer’s instructions. Microscopy was performed using a LSM 700 confocal microscope (Zeiss). Genotypes Fig. 1A-C ptc-GAL4,UAS-GFP/+ Fig. 1D-F ptc-GAL4,UAS-GFP/+; tubGAL80/UAS-Slik Fig. 1G-I ap-GAL4,UAS-GFP/slik Fig. 1J-L ptc-GAL4,UAS-GFP/+; tubGAL80/UAS-Slik Fig. 2C-D UAS-Dcr/+;ptc-GAL4, UASGFP/UAS-slikdsRNA Fig. 2E UAS-Dcr/+;ptc-GAL4, UAS-GFP/UASslikdsRNA;UAS-Slik/+ Fig. 2F UAS-Dcr/+;ptc-GAL4, UAS-GFP/UASslikdsRNA;UAS-Slik/+ Fig. 3A and K ptc-GAL4,UAS-GFP Fig. 3B ptc-GAL4,UAS-GFP/+; tubGAL80/UAS-Slik Fig. 3C ptc-GAL4,UAS-GFP/UAS-Dp110 Fig. 3D ptc-GAL4,UAS-GFP/+;UAS-dMyc/+ Fig. 3E ptc-GAL4,UAS-GFP/+;UASRas85D/+ Fig. 3F ptc-GAL4,UAS-GFP/+;UAS-CycD,UAScdk4/+ Fig. 3G ptc-GAL4,UAS-GFP/+;UAS-E2F,UASDP/+ Fig. 3H ptc-GAL4,UAS-GFP/+;ban/+ Fig. 3I ptc-GAL4,UAS-GFP/UAS-Ft Fig. 3J ptc-GAL4,UAS-GFP/egr Fig. 3L ptc-GAL4,UAS-GFP/+;hid/+ Fig. 4B ptc-GAL4,UAS-GFP/+ Fig. 4C ptc-GAL4/+;UAS-Slik/+ Fig. 4D ptc-GAL4,UAS-GFP/UAS-Slik Fig. 4E ptc-GAL4/+;UAS-Slik/+ Fig. 4F ptc-GAL4,UAS-GFP/+;UAS-Slik/+ Fig. 4H ptc-GAL4,UAS-GFP/+ Fig. 4I UAS-Slik/+;ptc-GAL4/+ Fig. 4J ptc-GAL4/+; UAS-Slik Fig. 4K ptc-GAL4/+; UAS-Slik Fig. 6C nub-GAL4/UAS-GFP Fig. 6D nub-GAL4/UAS-SlikdsRNA Fig. 6E nub-GAL4/UAS-slikdsRNA;UASSlik/+ Fig. 6F nub-GAL4/UAS-slikdsRNA;UASSlik/+ Fig. 6G nub-GAL4/UAS-slikdsRNA;UASSlik/+ Fig. 7A ptc-GAL4/UAS-slikdsRNA;UASSlik/+ Fig. 7B ptc-GAL4/UAS-slikdsRNA;UASSlik/+ Fig. 8A ap-GAL4/slik Fig. 8B ap-GAL4/+;UAS-Mer/+ Fig. 8C ap-GAL4/+;UAS-mer/+ Protein digestion Gel pieces were washed with water for 5 min and destained twice with the destaining buffer (50 mM ammonium bicarbonate, acetonitrile) for 15 min. An extra wash of 5 min was performed after destaining with a buffer of ammonium bicarbonate (50 mM). Gel pieces were then dehydrated with acetonitrile. Proteins were reduced by adding the reduction buffer (10 mM DTT, 100 mM ammonium bicarbonate) for 30 min at 40oC, and then alkylated by adding the alkylation buffer (55 mM iodoacetamide, 100 mM ammonium bicarbonate) for 20 min at 40oC. Gel pieces were dehydrated and washed at 40oC by adding ACN for 5 min before discarding all the reagents. Gel pieces were dried for 5 min at 40oC and then re-hydrated at 4oC for 40 min with enzyme solution. Tryptic digestion was performed with a 6 μg/ml solution of sequencing grade trypsin from Promega in 25 mM ammonium bicarbonate buffer, incubated at 58oC for 1 h and stopped with 15 μl of 1% formic acid/2% acetonitrile. Supernatant was transferred into a 96well plate and peptide extraction was performed with two 30-min extraction steps at room temperature using the extraction buffer (1% formic acid/50% ACN). All peptide extracts were pooled into the 96-well plate and then completely dried in vacuum centrifuge. The plate was sealed and stored at -20oC until LC-MS/MS analysis. LC-MS/MS analysis Prior to LC-MS/MS, protein digests were re-solubilized under agitation for 15 min in 21 μL of 1%ACN / 1% formic acid. The LC column was a PicoFrit fused silica capillary column (17 cm x 75 μm i.d; New Objective, Woburn, MA), self-packed with C-18 reverse-phase material (Jupiter 5 μm particles, 300 Å pore size; Phenomenex, Torrance, CA) using a high pressure packing cell. This column was installed on the Easy-nLC II system (Proxeon Biosystems, Odense, Denmark) and coupled to the by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of Slik by activation segment phosphorylation 6 LTQ Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) equipped with a Proxeon nanoelectrospray Flex ion source. The buffers used for chromatography were 0.2% formic acid (buffer A) and 100% acetonitrile/0.2% formic acid (buffer B). Peptides were loaded on-column at a flowrate of 600 nL/min and eluted with a 2 slope gradient at a flowrate of 250 nL/min. Solvent B first increased from 1 to 35% in 49 min and then from 35 to 80% B in 11 min. LC-MS/MS data acquisition was acquired using a data-dependant top10 method combined with a MS scanning upon detection of a neutral loss of phosphoric acid (48.99, 32.66 or 24.5 Th) in MS scans. The mass resolution for full MS scan was set to 60,000 (at m/z 400) and lock masses were used to improve mass accuracy. Mass over charge ratio range was from 375 to 1800 for MS scanning with a target value of 1,000,000 charges and from ~1/3 of parent m/z ratio to 1800 for MS scanning in the linear ion trap analyzer with a target value of 10,000 charges. The data dependent scan events used a maximum ion fill time of 100 ms and target ions already selected for MS/MS were dynamically excluded for 30 s after 2 repeat counts. Nanospray and S-lens voltages were set to 1.5 kV and 50 V, respectively. The normalized collision energy used was of 27 with an activation q of 0.25 and activation time of 10 ms. Capillary temperature was 250oC. Protein identification Protein database searches were performed using Mascot 2.3 (Matrix Science). The mass tolerance for precursor ions was set to 10 ppm and for fragment ions to 0.5 Da. The enzyme specified was trypsin and two missed cleavages were allowed. Cysteine carbamidomethylation was specified as a fixed modification, and methionine oxidation, serine, threonine and tyrosine phosphorylation as variable modifications. RESULTS The imaginal discs in Drosophila are a series of epithelial sacs that give rise to the adult appendages and body wall. Initially formed from invaginations of up to 50 cells in the embryonic epidermis, the discs grow rapidly during the four days of larval life to reach up to ~50,000 cells. The discs are divided into two continuous but morphologically distinct epithelial monolayers with their apical domains apposed and enclosing a central lumen. On one side of the lumen is the disc proper (DP) composed of pseudostratified columnar epithelial cells that will make most of the adult structures. DAPI staining of nuclei reveals the tight packing of DP cells (Fig. 1A). On the other side is the peripodial membrane (PM), made up of large flattened squamous cells with distinctly-spaced nuclei (Fig. 1A). This difference in nuclear spacing makes it easy to distinguish the two layers both in XY (Fig. 1A) and XZ (Fig. 1B) confocal optical sections. EdU incorporation assays reveal that DP cells are highly proliferative, whereas PM cells are not (Fig. 1C). Transgenic expression of wild-type Slik in DP cells using any one of several different GAL4 drivers (e.g. patched (ptc)-GAL4, apterous (ap)GAL4, nubbin-GAL4) has a striking nonautonomous effect on the PM cells (Fig. 1D and G, and not shown) (21). For example, in discs where ptc-GAL4 was used to drive expression of Slik in a central stripe of DP cells (marked by coexpression of GFP) (Fig. 1D), a large and abnormal cluster of densely packed PM cells appeared in a position directly overlying the Slikexpressing DP cells, as if these PM cells were responding to a signal from the Slik-expressing cells by proliferating. EdU incorporation assays confirmed that the PM cells within these clusters were rapidly proliferating (Fig. 1E), unlike normal PM cells (Fig. 1C). In Z-sections, it was clear that this cluster of proliferating cells was indeed in the PM and distinct from the DP, and that the GAL4 driver was not active in these cells (based on expression of GFP) (Fig. 1F). Slik expression using an independent GAL4 driver, ap-GAL4, which drives expression throughout the dorsal compartment of the DP (marked by co-expression of GFP), led to the appearance of a similar dense cluster of overproliferating PM cells overlying the dorsal compartment (Fig. 1G-I). As we previously reported (21), the same non-autonomous effect was observed when expressing a form of Slik (Slik) with the critical Asp residue involved in binding the catalytic magnesium ion (Asp) mutated to Asn, which is expected to disrupt catalytic activity (Fig. 1J-L). To confirm that this pro-proliferative effect is independent of Slik kinase activity, we tested the catalytic activity of Slik in cells and in vitro, using Moesin phosphorylation as a readout (14). by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of Slik by activation segment phosphorylation 7 Treatment of S2 cells with a dsRNA targeting the Slik 5′-untranslated region (UTR) efficiently depleted Slik protein, and led to a strong reduction of Moesin Thr phosphorylation in the cells (detected using a phosphospecific antiserum) (Fig. 2A). Transfection of a wild-type slik transgene lacking the 5′-UTR into slik 5′-UTR dsRNAtreated cells restored Moesin phosphorylation, whereas the comparable slik mutant did not (Fig. 2A). In in vitro kinase assays using [γ-P]ATP and kinase immunoprecipitated from transfected S2 cells, wild-type Slik phosphorylated a truncated form of Moesin consisting of the C-terminal actinbinding domain (Moe.CT) (Fig. 2B). Phosphorylation of Moe.CT was abolished by mutation of the critical regulatory Thr (Thr in Moesin) to Ala, confirming that Slik specifically phosphorylates this residue (Fig. 2B). In the same assay, Slik did not show any activity (Fig. 2B). We conclude that Slik does indeed lack catalytic activity. Catalytically inactive mutant forms of some kinases can dimerize with and activate wild-type kinases for example, catalytically inactive BRAF can activate CRAF in heterodimers (30). To rule out the possibility that Slik promotes proliferation by activating the wild-type kinase through dimerization, we expressed it in wing discs depleted of endogenous Slik. ptc-GAL4driven expression of a slik 5′-UTR dsRNA transgene efficiently depleted endogenous Slik (Fig. 2C), and did not affect PM cell proliferation (Fig. 2D). In this background, re-introduction of Slik had a similar effect as wild-type Slik in promoting non-autonomous proliferation of PM cells (Fig. 2E-F). Taken together, these results confirm that the ability of Slik to drive proliferation does not require catalytic activity. Slik specifically stimulates non-autonomous proliferation Slik expression has pleiotropic effects in discs, accelerating cell proliferation rates and tissue growth while also increasing apoptosis (21). Altering these processes can have compensatory non-autonomous effects on tissue growth (31,32). To see if the non-autonomous proproliferative effects of Slik could be an indirect consequence of its effect on growth and cell survival, we tested whether expression of other genes that affect these processes in DP cells could have a similar effect on PM morphology. Genes whose expression accelerates primarily cell growth, such as the phosphatidylinositol-3-kinase catalytic subunit Dp110 (33), Myc (34), and activated Ras85D (35) had strong cell autonomous effects in DP cells (as evidenced by an obvious increase in space between nuclei within the expression domain) but did not lead to the appearance of a cluster of PM cell nuclei as Slik did (Fig. 3A-E). CyclinD and Cdk4, which together promote cell growth in Drosophila (36), also did not noticeably alter PM morphology (Fig. 3F). Accelerating cell division rates by coexpressing the cell cycle regulator E2F and its cofactor DP (37) led to the expected autonomous reduction in DP cell size (and hence decreased nuclear spacing) without affecting PM morphology (Fig. 3G). Manipulations that coordinately accelerate cell proliferation and tissue growth, such as expression of the miRNA bantam (38) or expression of a dominant negative form of the tumour suppressor Fat (Fat) (39), induced robust overgrowth in adult wings (data not shown) but did not noticeably alter PM morphology (Fig. 3H-I). Finally, induction of apoptosis by expressing either the Tumour necrosis factor ligand Eiger (40) or the pro-apoptotic protein Hid (41), which had obvious effects on DP morphology (Eiger) and DP cell survival (Hid), did not noticeably alter PM morphology (Fig. 3JL). The fact that each of these regulators produced robust cell autonomous effects in DP cells without affecting PM morphology strongly suggests that the non-autonomous effect of Slik on cell proliferation is the result of Slik signaling rather that an indirect consequence of altered proliferation or apoptosis. Slik-driven tissue growth, but not Moesin phosphorylation, depends on proper localization In immunostainings, Slik is diffusely localized throughout imaginal disc cells and is concentrated apically, where the majority of P-Moe staining is observed (21). To see which of the three domains of Slik (kinase, NCD, or CCD) (Fig. 4A) might mediate this localization, we generated and expressed transgenes encoding the domains either individually or in various combinations. A Myctagged full-length form of Slik recapitulated the normal localization pattern (Fig. 4B and C). We previously reported that expression of just the kinase domain (Slik) caused a redistribution of by gest on N ovem er 8, 2017 hp://w w w .jb.org/ D ow nladed from Regulation of Slik by activation segment phosphorylation 8 P-Moe away from the apical domain in wing disc cells (21), implying that localization of the kinase was affected in the absence of the linker and/or CCD. In fact, Slik accumulated primarily in the nucleus of imaginal disc cells, as did a form of Slik lacking just the CCD (Slik) (Fig. 4D-E). Conversely, the CCD of Slik was sufficient for apical localization (Fig. 4F). The comparable domain of mammalian LOK mediates apical microvilli localization of the kinase in cultured cells, suggesting it is a conserved localization domain (16). Domains outside of the kinase domain of some protein kinases are responsible for their noncatalytic activites (6). For example, the regulatory domain of c-Raf alone is sufficient to mediate cRaf-dependent inhibition of Mst2 (42). Therefore, we hypothesized that the CCD might not only localize Slik in cells but also directly mediate its growth-promoting activity. To test this, we examined the requirement for the CCD in Slik function. The CCD was dispensable for Moesin phosphorylation in S2 cells (Fig. 4G), consistent with the in vivo effects of Slik expression on PMoe re-distribution referred to above (21). In contrast, removal of the CCD abrogated the effect of Slik on proliferation (Fig. 4H-J). Although required, the CCD alone was not sufficient to stimulate proliferation (Fig. 4K). Together these results imply that multiple regions of the protein are required for non-catalytic signaling. We conclude that the CCD is important for localizing Slik within cells, restricting Moesin phosphorylation to the appropriate apical site and positioning the protein to activate pro-proliferative signaling. Moesin activation occurs at sites of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) accumulation, as its phosphorylation is dependent upon prior binding to PI(4,5)P2 (43). To see if this might also be involved in membrane recruitment of Slik, we tested the ability of the CCD to bind to phosphoiniositides. In pulldowns of cell lysates from S2 cells expressing Slik, we did not detect interaction of the CCD with any phosphoinositides (Fig. 4L). Pulldown of the PI(4,5)P2-binding PH domain of PLC-δ from S2 cell lysates using PI(4,5)P2-coupled beads confirmed that the assay was working (Fig. 4L). We conclude that apical localization of Slik likely does not involve direct interaction of the CCD with membrane