Regulation of Catalytic and Non-catalytic Functions of the Drosophila Ste20 Kinase Slik by Activation Segment Phosphorylation*

Background: Slik kinase has catalytic activity-dependent and -independent functions. Results: Mutation of activation segment phosphorylation sites abolishes both catalytic and non-catalytic activities; non-catalytic function also depends upon localization via the C-terminal domain. Conclusion: Slik is regulated by both localization and phosphorylation. Significance: Conformational activation can control not only catalytic but also non-catalytic activities of kinases. Protein kinases carry out important functions in cells both by phosphorylating substrates and by means of regulated non-catalytic 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 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 auto- and 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 C-terminal coiled-coil domain 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 carry out important functions in cells both by phosphorylating substrates and by means of regulated non-catalytic 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 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 auto-and 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 C-terminal coiled-coil domain and activation via activation segment phosphorylation, which enhances both phosphorylation of substrates like Moesin and engagement of effectors of its noncatalytic 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) 3 kinases are a large group of Ser/Thr kinases with 28 members in humans divided into 2 distinct families (PAK-and GCK-like) and 10 subfamilies. Outside of the catalytic domain, which shows homology to the yeast kinase Ste20p, the 10 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 tumor suppression (4), and the GCK-VI subfamily kinases Osr1 and Spak in regulating ion channels (5). Many others are less well characterized. Although 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 Ste20-like kinase domain and a C-terminal coiledcoiled repeat-containing domain (CCD) connected by a nonconserved 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 cross-linkers 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 N-and C-terminal 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 microvilli (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)(23)(24)(25)(26)(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). To understand how these distinct activities of Slik may be regulated, we undertook a structure-function 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 growth-promoting (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 full-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 T186A , Slik T192A , and Slik T186A/T192A mutants, the first ϳ700 bp of the modified 5Ј slik coding sequence were PCRamplified 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 C terminus 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 T196A mutant, an EcoRI/NarI fragment was used instead because the KpnI site is destroyed by the introduced mutation. The kinase dead Slik kd mutant, in which Asp 176 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 T558A and Moesin T558D 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 C terminus of Moesin (amino acids 311-575 of Moe-PD) in pET-23b. For the Slik domain constructs, sequences coding for amino acids 1-321 (Slik kin ), 322-988 (Slik NCD ), or 989 -1300 (Slik CCD ) of Slik were PCRamplified with primers that introduced a 5Ј EcoRI site and 3Ј KpnI site. EcoRI-and KpnI-digested products were cloned into pMT.puro3c and pUAST plasmids modified to encode add an N-terminal Myc epitope tag. To generate the slik 5Ј-untranslated region (UTR) dsRNA transgene, the UTR sequence (spanning three exons between nucleotides 24,401,370 and 24,402,688 of the Drosophila chromosome 2R scaffold) was PCR-amplified 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 5Ј-UTR dsRNA for cell culture experiments. A plasmid encoding PLC␦-PH fused to GFP was generously provided by Dr. J. Brill and was re-cloned into pMT.puro3c for expression in S2 cells.
Cell Culture-slik 5Ј-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 95°C for 5 min, and then slowly cooled to room temperature. All cell-based experiments were done in Drosophila S2-Rϩ cells grown in Schneider's medium (Lonza) supplemented with 10% FBS (Gibco) and 50 units/ml penicillin/streptomycin (Gibco) at 25°C. Cells were plated at 1 ϫ 10 6 / ml. For RNA interference, the appropriate dsRNA was added to the culture medium on day 0 at 1 g/ml and left to incubate for 48 h. Plasmids were then transfected on day 2 using the X-tremeGENE HP reagent (Roche Applied Science) as described by the manufacturer. Typically, 500 ng/ml Slik plasmids and 200 ng/ml Moesin plasmids were added. On day 3, an additional dose of dsRNA was added along with 0.5 mM CuSO 4 for induction. The cells were finally processed on day 5.
Immunoprecipitations, Pulldown Assays, and Western Blotting-Cells were harvested in ice-cold PBS and then lysed for 15mins at 4°C in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40 with added 1ϫ protease inhibitor (Roche Applied Science), 1ϫ AEBSF (Sigma), and 1ϫ phosphatase inhibitor (Roche Applied Science)). Lysates were centrifuged to remove cell debris (12,000 ϫ g, 15 min, 4°C). For immunoprecipitations, cleared lysates were incubated with the appropriate antibody for 1 h at 4°C and then with protein A/G-agarose beads (Santa Cruz) for 1 h at 4°C. The beads were washed in lysis buffer 3 times for 5 min and then suspended in 2ϫ Laemmli sample buffer at 80°C for 5 min 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 pulldown assays, cleared lysates from cells expressing either Slik CCD or the PH domain of phospholipase C␦ were incubated with agarose beads coupled to various phosphatidylinositol phosphates according to the manufacturer's instructions (Echelon Biosciences), and bound proteins were 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 Escherichia coli BL21 cells and purified using glutathione-agarose (Pierce) according to the manufacturer's protocol. S2 cells were transfected with Slik or Slik kd expression vectors and induced as described above. Three days after induction, cells were washed with PBS and lysed for 15 min on ice in immunoprecipitation lysis buffer (25 mM HEPES, pH 7.2, 100 mM NaCl, 10% glycerol, 0.5% Nonidet P-40 containing 5 mM EGTA, 5 mM EDTA, 50 mM NaF, 10 mM ␤-glycerophosphate, 1 mM NaVO4, 0.5 mg/ml AEBSF, and 1ϫ PhosSTOP (Roche)). 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 were incubated an additional 2 h. The beads were washed 3 times at 4°C in 25 mM HEPES, pH 7.2, 150 mM NaCl, 0.5% Nonidet P-40 containing 5 mM EGTA, 5 mM EDTA, 10 mM ␤-glycerophosphate, 1 mM NaVO 4 , 1ϫ PhosSTOP, and 0.01% BSA. Beads were rinsed two times in kinase buffer (20 mM Tris, pH 7.5, 15 mM MgCl 2 , 3 mM MnCl2, 1 mM DTT, 1 mM NaVO 4 , 10 mM ␤-glycerophosphate, 0.1 mg/ml AEBSF, 1ϫ PhosSTOP) and then suspended in kinase buffer containing 50 M ATP, 5 Ci [␥-32 P]ATP (for radioactive labeling), and 5 l of GST-Moe.CT substrate. Reactions were incubated for 20 min at 30°C, stopped by the addition of 5ϫ 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 ts was used to inhibit transgene expression during embryonic stages. Parents were allowed to lay eggs at 21°C for 2 days. One day later, offspring were transferred to 27°C to inhibit GAL80 ts 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% paraformaldehyde in 0.2% PBS, 0.2% Tween (PBT) for 20 min. The anterior halves were then washed 3 times in 0.2% PBT followed by blocking for 1 h in BBT (0.2% PBT ϩ 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 h with the fluorescent secondary antibodies diluted in BBT (Invitrogen or Jackson ImmunoResearch Laboratories, Inc.). Samples were again washed 3 times in 0.2% PBT and then incubated for 10 min with 2.5 g/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 were dissected in serum-free medium (EX-CELL 420, Sigma), and cleaned anterior halves were labeled for 1 h with EdU and processed using the Click-iT Edu Alexa Fluor 55 Imaging Kit (Life Technologies) according to the manufacturer's instructions. Microscopy was performed using a LSM 700 confocal microscope (Zeiss).
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 40°C and then alkylated by adding the alkylation buffer (55 mM iodoacetamide, 100 mM ammonium bicarbonate) for 20 min at 40°C. Gel pieces were dehydrated and washed at 40°C by adding acetonitrile for 5 min before discarding all the reagents. Gel pieces were dried for 5 min at 40°C and then re-hydrated at 4°C for 40 min with enzyme solution. Tryptic digestion was performed with a 6 g/ml solution of sequencing grade trypsin from Promega in 25 mM ammonium bicarbonate buffer, incubated at 58°C for 1 h, and stopped with 15 l of 1% formic acid, 2% acetonitrile. Supernatant was transferred into a 96-well plate, and peptide extraction was performed with two 30-min extraction steps at room temperature using the extraction buffer (1% formic acid, 50% acetonitrile). All peptide extracts were pooled into the 96-well plate and then completely dried in a vacuum centrifuge. The plate was sealed and stored at Ϫ20°C until liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis.
LC-MS/MS Analysis-Before LC-MS/MS, protein digests were re-solubilized under agitation for 15 min in 21 l of 1% acetonitrile, 1% formic acid. The LC column was a PicoFrit fused silica capillary column (17 cm ϫ 75 m inner diameter; New Objective, Woburn, MA) self-packed with C-18 reversephase material (Jupiter 5 m 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 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 flow rate of 600 nl/min and eluted with a 2 slope gradient at a flow rate 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-dependent top 10 method combined with a MS 3 scanning upon detection of a neutral loss of phosphoric acid (48.99, 32.66, or 24.5 thomsons) in MS 2 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 1,800 for MS scanning with a target value of 1,000,000 charges and from ϳ 1 ⁄ 3 of parent m/z ratio to 1,800 for MS n 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 250°C.
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 that 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 were specified 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 4 days of larval life to reach up to ϳ50,000 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 non-autonomous effect on the PM cells (Fig. 1, D 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 co-expression of GFP) (Fig. 1D), a large and abnormal cluster of densely packed PM cells appeared in a position directly overlying the Slik-expressing 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. 1, G-I).
As we previously reported (21), the same non-autonomous effect was observed when expressing a form of Slik (Slik kd ) with the critical Asp residue involved in binding the catalytic magnesium ion (Asp 176 ) mutated to Asn, which is expected to disrupt catalytic activity (Fig. 1, J-L). To confirm that this proproliferative effect is independent of Slik kinase activity, we tested the catalytic activity of Slik kd in cells and in vitro using Moesin phosphorylation as a readout (14). Treatment of S2 cells with a dsRNA targeting the Slik 5Ј-untranslated region (UTR) efficiently depleted Slik protein and led to a strong reduction of Moesin Thr 556 phosphorylation in the cells (detected using a phosphospecific antiserum) ( Fig. 2A). Transfection of a wild-type slik transgene lacking the 5Ј-UTR into slik 5Ј-UTR dsRNA-treated cells restored Moesin phosphorylation, whereas the comparable slik kd mutant did not ( Fig. 2A). In in vitro kinase assays using [␥-32 P]ATP and kinase immunoprecipitated from transfected S2 cells, wild-type Slik phosphorylated a truncated form of Moesin consisting of the C-terminal actin-binding domain (Moe.CT) (Fig. 2B). Phosphorylation of Moe.CT was abolished by mutation of the critical regulatory Thr (Thr 556 in Moesin) to Ala, confirming that Slik specifically phosphorylates this residue (Fig. 2B). In the same assay, Slik kd did not show any activity (Fig. 2B). We conclude that Slik kd 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 kd promotes proliferation by activating the wild-type kinase through dimerization, we expressed it in wing discs depleted of endogenous Slik. ptc-GAL4-driven expression of a slik 5Ј-UTR dsRNA transgene efficiently depleted endogenous Slik (Fig. 2C) and did not affect PM cell proliferation (Fig. 2D). In this background, reintroduction of Slik kd had a similar effect as wild-type Slik in promoting non-autonomous proliferation of PM cells (Fig. 2, E and 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 pro-proliferative 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 did Slik (Fig. 3, A-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 co-expressing the cell cycle regulator E2F and its co-factor 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 tumor suppressor Fat (Fat ⌬icd ) (39), induced robust overgrowth in adult wings (data not shown) but did not noticeably alter PM morphology (Fig. 3, H-I). Finally, induction of apoptosis by expressing either the tumor 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. 3, J-L). 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 Myc-tagged full-length form of Slik recapitulated the normal localization pattern (Fig. 4, B and C). We previously reported that expression of just the kinase domain (Slik kin ) caused a redistribution of 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 kin accumulated primarily in the nucleus of imaginal disc cells, as did a form of Slik lacking just the CCD (Slik ⌬CCD ) (Fig. 4, D and 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 non-catalytic activities (6). For example, the regulatory domain of c-Raf alone is sufficient to mediate c-Raf-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 kin expression on P-Moe re-distribution referred to above (21). In contrast, removal of the CCD abrogated the effect of Slik on proliferation (Fig. 4, H-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-4,5bisphosphate (PI(4,5)P 2 ) accumulation, as its phosphorylation is dependent upon prior binding to PI(4,5)P 2 (43). To see if this might also be involved in membrane recruitment of Slik, we tested the ability of the CCD to bind to phosphoinositides. In pulldown assays of cell lysates from S2 cells expressing Slik CCD , we did not detect interaction of the CCD with any phosphoinositides (Fig. 4L). Pulldown of the PI(4,5)P 2 binding PH domain of PLC-␦ from S2 cell lysates using PI(4,5)P 2 -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 phosphoinositides.
Regulation of Slik Catalytic Activity by Phosphorylation-Both in wing discs and in S2 cells, Slik is a phosphoprotein, as evidenced by an increase in mobility in SDS-PAGE gels after treatment of lysates with -phosphatase (Fig. 5A). At least some of this is likely attributable to autophosphorylation, as Slik is capable of phosphorylating itself in in vitro kinase assays (Fig.  5B). To see if Slik may also be a substrate of other kinases, we compared the phosphorylation status of transfected Slik and Slik kd in S2 cells depleted of endogenous Slik. As expected, transfected Slik shifted in response to -phosphatase treatment (Fig. 5C). Interestingly, we observed a very similar shift of Slik kd in response to -phosphatase treatment (Fig. 5C). This suggests that Slik is also phosphorylated and potentially regulated by other kinases.
We used a LC-MS/MS approach to map phosphorylated residues in Slik. We carried out this analysis on Slik protein isolated from transfected S2 cells and on endogenous Slik isolated from third instar larvae. All 19 of the phosphosites we identified (Table 1) mapped to either the kinase domain or NCD, with 15 of them observed in both S2 cells and embryos (Fig. 5D). Two clusters of phosphorylation sites, one in the activation segment (Thr 186 , Thr 192 , Thr 196 ) and one just beyond the C-terminal end of the kinase domain (Ser 340 , Ser 342 , Ser 345 , and Ser 354 ), stood out, with both corresponding to conserved phosphorylation sites that have been observed in LC-MS/MS analyses of the mammalian orthologues Slk and Lok (according to the Phosphosite Plus database (44)).
The activation segment is a common site of regulatory phosphorylation in most eukaryotic protein kinases. This extended polypeptide region, situated between the highly conserved Asp-Phe-Gly (DFG) and Ala-Pro-Glu (APE) tripeptide motifs, wraps across the surface of the large lobe of the kinase domain. In its unphosphorylated state, the activation segment is highly dynamic and disordered. Phosphorylation at one or more residues in this region stabilizes the kinase in its active conformation, with the so-called contiguous "hydrophobic spine" (regulatory or R-spine) at the kinase domain core, composed of residues from both the small (N-) and large (C-) lobes, properly aligned. This is generally required for efficient catalysis (for review, see Refs. 45 and 46). Ste20 kinases are typical in this respect, with activation segment phosphorylation being crucial for the activity of several (47)(48)(49)(50).
Phosphorylation at three Ser/Thr residues in the activation segments of Slk and Lok has been observed numerous times (Ն48 occurrences at each site for the two proteins combined) in independent LC-MS/MS analyses (44). Phosphorylation at two of these sites, Thr 183 and Ser 189 of human Slk (corresponding to Thr 186 and Thr 192 in Slik), was observed in a crystal structure of the kinase domain of this protein (51), and mutational analysis indicated that both phospho-residues are important for activation of its catalytic activity toward an exogenous substrate (52). Mutation of the third site, Thr 193 (corresponding to Thr 196 in Slik), impaired catalytic activity of the isolated kinase domain of Slk (52). Consistent with this, we found that mutating both Thr 186 and Thr 192 in Slik to Ala (Slik T186A/T192A ) strongly impaired its ability to rescue Moesin phosphorylation in endogenous Slik-depleted cells (Fig. 6A). Phosphorylation at either site was sufficient for at least some activity, as both single mutants either fully (Slik T186A ) or partially (Slik T192A ) rescued Moesin phosphorylation (Fig. 6A). We observed the same trend in in vitro kinase assays (Fig. 6B). In contrast, mutation of the single site Thr 196 to Ala (Slik T196A ) strongly impaired catalytic activity both in cells and in vitro (Fig. 6A and B).
To carry out similar tests in vivo, we generated transgenic flies for expressing the wild-type and mutant forms of Slik. All transgenes were recombined into the same site in the genome using the C31-based integration system (29) to ensure equal mRNA expression. We used nub-GAL4 to drive expression of the slik 5Ј-UTR RNAi transgene, which efficiently depleted endogenous Slik protein and caused a strong reduction of Moesin phosphorylation specifically throughout the wing pouch of developing wing discs, as compared with controls (Fig. 6, C and  D). Co-expression of wild-type Slik increased Moesin phosphorylation to well above endogenous levels (Fig. 6E). Co-expression of either Slik T186A/T192A or Slik T196A partially rescued Moesin phosphorylation but only to about endogenous levels (Fig. 6, F  and G), confirming that both mutants have impaired activity. Taken together, our results suggest that phosphorylation of Slik at Thr 196 and either Thr 186 or Thr 192 is required for efficient phosphorylation of its substrates. We were unable to detect any difference in the subcellular distribution of the activation segment mutants versus wild-type Slik (Fig. 6, E-G, insets), suggesting that apical localization occurs independently of activation.
The Non-catalytic Function of Slik in Growth Control Is Dependent upon Kinase Activation-Because Slik can promote tissue growth independently of substrate phosphorylation, it remained an open question whether this activity is regulated in any way. To test whether activation loop phosphorylation could play a role in this regard, we used the rescue assay to assess the ability of activation loop mutants of Slik to drive non-autonomous proliferation. Expression of wild-type Slik or Slik D176N with ptc-GAL4 caused fully penetrant lethality, with many animals dying at pupal stage. In contrast, Slik T186A/T192A and Slik T196A expression was much better tolerated, with some or all animals surviving to adult stage, respectively (data not shown). Interestingly, whereas expression of wild-type Slik or Slik D176N in endogenous Slik-depleted DP cells triggered the expected non-autonomous proliferation effect (Fig. 2, E and F), Slik T186A/T192A did so much more weakly and Slik T196A not at all (Fig. 7, A and B). Thus, although it does not require catalytic activity per se, the ability of Slik to induce non-autonomous proliferation in discs does require that the kinase be "activated" by activation segment phosphorylation, providing a level of regulation.
Slik has been linked to regulation of the tumor suppressor protein Mer/NF2 which, like Moesin, is a FERM domain-containing protein. Based on work in both flies and mammals, Mer/ NF-2 mediates its growth-suppressive effects by activating the Hippo (Hpo)/Mst pathway (53)(54)(55)(56). Like Moesin, Mer/NF2 exists in active and inactive conformations, with the transition from active to inactive states driven by phosphorylation at a critical residue (Ser 518 in human NF2) (57). Manipulating Slik activity altered Mer phosphorylation, which led to the suggestion that Slik promotes growth by phosphorylating and inhibiting Mer (58). This is hard to reconcile with the observation that Slik promotes tissue growth independently of its catalytic activity. Furthermore, the residue in Mer/NF2 corresponding to the critical Thr in Moesin that is phosphorylated by Slik (Thr 576 in human NF2) does not appear to be involved in regulating its activity (59).
To see if inhibition of Mer by Slik could be sufficient to explain its effects on growth, we compared the phenotypic effects of expressing Slik or inhibiting Mer in discs. ap-GAL4driven expression of Slik in dorsal DP cells induced robust nonautonomous proliferation of PM cells directly overlying the DP dorsal compartment (Fig. 8A). In contrast, under the same conditions neither expression of a dominant negative form of Mer (Mer ⌬BB ) (60) nor mer dsRNA had nonautonomous effects on PM cell proliferation (Fig. 8, B and C), although both induced robust overgrowth of the dorsal wing surface that caused the wings to curve downward (Fig. 8, D and E, and not shown). Taken together, our results suggest that Mer is not the main target of Slik in regulating tissue growth.

Discussion
The Drosophila Ste20 kinase Slik is one of a growing number of kinases with both catalytic activity-dependent and -independent functions (6). The non-catalytic functions of kinases are mediated in different ways, including scaffolding of protein FIGURE 5. Slik is a phosphoprotein in vivo. Western blot of lysates from S2 cells or third-instar wing discs treated without (Ϫ) or with (ϩ) -phosphatase, probed with anti-Slik antibody, reveals that Slik is a phosphoprotein in cells and in tissues. B, in vitro kinase assay using Slik or Slik kd immunoprecipitated from S2 cells. 32 P-Labeling indicates that wild-type Slik but not Slik kd is capable of autophosphorylation. C, Western blot of lysates from S2 cells treated without (Ϫ) or with (ϩ) dsRNA targeting the 5Ј-UTR of slik and transfected with empty vector (Ϫ) or transgenes encoding Slik or Slik kd . Depletion of endogenous Slik was efficient. Despite being unable to autophosphorylate, Slik kd shows a similar phosphorylation shift as wild-type Slik after -phosphatase treatment, suggesting that Slik is phosphorylated by other kinases. D, summary of phosphorylation site mapping by LC-MS/MS. Sites in yellow were identified in Slik isolated from S2 cells, sites in blue were identified in Slik from wing discs, and sites in green were identified in both. Sites identified more than five times in the Phosphosite database for mammalian Slk are indicated below.
complexes, allosteric regulation of other proteins, and competition for binding partners. We were interested to know whether the regulatory mechanisms that control Slik catalytic activity, which is required for phosphorylation of the substrate Moesin and thus epithelial tissue integrity in vivo, also control its non-catalytic growth-promoting function. Our results indicate that activation segment phosphorylation, which typically stabilizes kinases in their active conformation, activates the catalytic activity of Slik and is also important for its non-catalytic function. In contrast, localization of the kinase via its conserved CCD is not required for catalytic activity but is essential for Slik to drive cell proliferation.
Analysis of the crystal structures of many protein kinase domains in the phosphorylated active and non-phosphorylated inactive states has revealed how activation segment phosphorylation affects kinase conformation and thus catalytic activity (45,46). In the structures of protein kinase A (PKA) and others, the primary activation segment phosphate group (on Thr 196 in PKA) stabilizes the active conformation by making electrostatic contacts with several conserved motifs in the kinase core. Some kinases also contain a secondary phosphorylation site, whose main function is less clear. Of the three activation segment residues we identified, Thr 192 in Slik aligns most closely with Thr 196 in PKA and is conserved (Ser or Thr) in all Ste20 kinases. In most of these proteins, this residue is the primary regulatory phosphorylation site in the activation loop (2). For some, this residue is autophosphorylated (e.g. Pak2 (61), hMink (62), Slk . Slik catalytic activity is regulated by activation segment phosphorylation. A, "in-cell" assay of Slik catalytic activity. Lysates from S2 cells depleted of endogenous Slik and transfected with empty vector (Ϫ) or transgenes encoding wild-type Slik or the indicated activation loop phosphorylation site mutants were analyzed by Western blotting. The Slik T186A mutant rescued Moesin phosphorylation to a similar extent as wild-type Slik, Slik T192A was somewhat less active, and the Slik T186A,T192A double mutant and Slik T196A had little activity in cells. B, in vitro kinase assay of Slik activity using the C-terminal actin binding domain of Moesin fused to GST (GST-Moe.CT) as a substrate. Slik T186A was fully active, and the activity of Slik T192A , Slik T186A,T192A , and Slik T196A was strongly impaired. C-G, confocal images of wing discs in which transgenes were expressed throughout the wing pouch using the nub-GAL4 driver and immunostained with antibodies against Slik (green) and phospho-Moesin (red). Slik and phospho-Moesin are normally ubiquitous throughout the discs, as seen in control discs with nub-GAL4-driven expression of GFP (blue) (C). nub-GAL4-driven expression of a dsRNA transgene targeting the 5Ј-UTR of slik efficiently depleted Slik protein and led to a strong reduction in Moesin phosphorylation (D). Re-introduction of wild-type Slik dramatically increased Moesin phosphorylation (E). Although they retained some activity, Slik T186A,T192A and Slik T196A were substantially less active (F and G). Insets in E-G show magnified views of Slik staining at a fold in the disc, where strong Slik accumulation in the apical region of cells on either side of the fold, optically sectioned longitudinally (arrowheads), is clearly visible for all three transgenic Slik variants. (51)). For others, such as Osr1 and Spak (50), Hippo (27), Pak1 (49), and Hpk1 (48), the site can also be trans-phosphorylated, providing a point of intersection with other signaling pathways.
The residue corresponding to Thr 186 is less well conserved, being present in Slk, Lok, and Hippo/Mst kinases but not in the PAK kinases or Osr1 and Spak. In those kinases where it is present, this site appears to function as a secondary phosphorylation site (2,46). Our results with Slik fit this model, as mutation of Thr 192 alone had more of an effect on catalytic activity than Thr 186 . Phosphorylation of at least one of these residues is important for efficient catalysis, as the double mutant had low activity. Both are likely autophosphorylated, as phosphate groups were observed at the equivalent sites in bacterially expressed recombinant Slk and Lok kinase domains (51). However, our genetic analyses suggest that at least one of these sites is likely also trans-phosphorylated (see below).
In addition to these two well defined sites, Slik activity was strongly impaired by mutation of Thr 196 . This residue is situated at a hinge point near the start of the "p ϩ 1" loop region of the activation segment, so-called because several residues in this region make contact with the p ϩ 1 residue in the substrate. The equivalent residue in other kinases also makes contact with conserved residues in the catalytic loop (46), meaning that modification of this residue could influence both the active structure and interaction with substrates. This Thr residue is invariant in all Ste20 kinases (2), and phosphorylation at this site appears to be fairly common (based on analysis of the Phosphosite MS/MS database (44)), especially for certain kinases (e.g. Slk and Lok, Spak and Osr1, and Ysk/Stk25). In several Ste20 kinases, including Slk, Spak, and Mst1, Ala substitution at this site impairs substrate phosphorylation (52,63). Thus the role of phosphorylation at this site in regulating kinase function is conserved both between at least some Ste20 kinase family members and between species. The mechanism remains to be determined.
Although there are few cases where the involvement of activation segment phosphorylation in regulating catalytic activityindependent activities of kinases has been assessed, there is evidence that it can either be required (as in activation of topo-II␣ and Parp-1 by ERKs (64, 65)) or can cause a switch from catalytic activity-independent to -dependent signaling (for yeast Kss1 (66)). We found that the catalytic activity-independent growth promoting function of Slik shows a similar dependence on activation loop phosphorylation as catalytic activity; Thr 196 and either Thr 186 or Thr 192 need to be phosphorylated for an efficient proliferative response. Although Slik can autophosphorylate, it is likely also trans-phosphorylated by one or more other kinases, as the wild-type and catalytically inactive proteins were phosphorylated to a similar extent in endogenous Slik-depleted cells. Interestingly, the catalytically inactive Slik kd mutant protein cannot autophosphorylate but is capable of promoting cell proliferation even when expressed in cells depleted of endogenous Slik. This suggests that the activation segment of Slik can be phosphorylated by another kinase to activate non-catalytic pro-proliferative signaling.
We identified 16 phosphorylation sites outside of the activation segment of Slik in our MS/MS analyses. The majority of these were in the non-conserved central domain, and their relevance is unclear. One cluster of sites just beyond the C-terminal limit of the kinase domain (Ser 340 , Ser 342 , Ser 345 , and Ser 354 ) appears to be conserved in spacing, if not in actual primary sequence, with phosphorylation sites in mammalian Slk and Lok. Interestingly, two of the corresponding sites in mammalian Slk (Ser 347 and Ser 348 ) mediate negative regulation by casein kinase II in response to v-Src activity (67), suggesting the possibility that these may be conserved trans-regulatory phosphorylation sites.
Consistent with studies of mammalian Slk (16), we found that the coiled-coil-containing CCD of Slik mediates apical localization of the protein in epithelial cells of the imaginal disc. Although a model involving membrane recruitment of Slik by PI(4,5)P 2 would have fit well with ERM protein biology, we did not detect any interaction of the CCD with phosphoinositol lipids. Interestingly, the CCD also appears to be involved in regulated membrane recruitment of Slk in non-polarized mouse embryonic fibroblasts through interactions with the LIM domain transcription cofactors Ldb1 and Ldb2 (68). Although we found that it determines where in cells Moesin gets activated, the CCD is not required for phosphorylation per se. In fact, previous work suggests that the Slk CCD acts as an autoinhibitory domain (68), something that we have not tested. In contrast, the CCD is absolutely required for activation of pro-proliferative signaling. Although non-catalytic activities of many kinases are mediated by parts of the protein outside of the kinase domain, the CCD alone is not capable of stimulating proliferation. Our data are more consistent with a model in which localization via the CCD instead brings Slik into the vicinity of other proteins required for signaling, and activation loop phosphorylation places Slik in a conformation that enables it to interact with these proteins to initiate the signal.
We do not know how Slik promotes cell proliferation in the wing disc. Although Slik in some way influences the phosphorylation of Mer, the importance of that for the Slik-driven growth phenotypes is not clear for two reasons. First, Slikdriven growth does not require catalytic activity. Second, although it causes the expected tissue overgrowth response, direct inhibition of Mer activity in wing discs does not induce non-autonomous proliferation, which is a characteristic effect of Slik. Thus, although Mer regulation may contribute to the growth effects of Slik, it does not appear to be the main target. The direct target of Slik in growth signaling remains to be identified.