Regulation of Polycystin-1 Function by Calmodulin Binding

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common genetic disease that leads to progressive renal cyst growth and loss of renal function, and is caused by mutations in the genes encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. The PC1/PC2 complex localizes to primary cilia and can act as a flow-dependent calcium channel in addition to numerous other signaling functions. The exact functions of the polycystins, their regulation and the purpose of the PC1/PC2 channel are still poorly understood. PC1 is an integral membrane protein with a large extracytoplasmic N-terminal domain and a short, ~200 amino acid C-terminal cytoplasmic tail. Most proteins that interact with PC1 have been found to bind via the cytoplasmic tail. Here we report that the PC1 tail has homology to the regulatory domain of myosin heavy chain including a conserved calmodulin-binding motif. This motif binds to CaM in a calcium-dependent manner. Disruption of the CaM-binding motif in PC1 does not affect PC2 binding, cilia targeting, or signaling via heterotrimeric G-proteins or STAT3. However, disruption of CaM binding inhibits the PC1/PC2 calcium channel activity and the flow-dependent calcium response in kidney epithelial cells. Furthermore, expression of CaM-binding mutant PC1 disrupts cellular energy metabolism. These results suggest that critical functions of PC1 are regulated by its ability to sense cytosolic calcium levels via binding to CaM.


Introduction
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a common, life-threatening genetic disease with an estimated prevalence between 1:400-1:1000 live births. The underlying causes are mutations in either the PKD1 or PKD2 genes encoding polycystin-1 (PC1) and putative structure of the PC1 tail, we performed a homology search against proteins with known structural information in the Protein Data Bank (www.rcsb.org [30]). Pairwise alignments led to the identification of a portion of the PC1 tail (residues 4100-4143) with significant homology to the regulatory domain of scallop myosin (Fig 1A).
This region of myosin heavy chains from many species typically contains two tandem IQ motifs that form binding sites for CaM or the CaM-related proteins myosin essential light chain (ELC) and myosin regulatory light chain (RLC), respectively [31][32][33]. An extended sequence comparison between PC1 and myosin heavy chains from a variety of divergent species shows that while PC1 is clearly distinct from myosins, some of the most conserved myosin residues are also conserved in PC1 (S1 Fig).
Based on the primary sequence alignment and the known 3D-structure of scallop myosin in complex with ELC and RLC [31], we modeled the predicted structure of the corresponding region of the PC1 tail which resulted in a relatively extended, S-shaped alpha helix (Fig 1B).
Given this putative structural homology, we hypothesized that this region of PC1 may interact with CaM. The PC1 tail sequence was analyzed for possible CaM binding motifs using a prediction algorithm (Calmodulin Target Database) [34,35]. Indeed the algorithm predicts two CaM binding motifs that overlap with the regions of homology to the ELC and RLC binding motifs in myosin heavy chain ( Fig 1C). However, PC1 does not contain a classical IQ motif as it lacks the obligate glutamines in the second position. Instead, the first putative CaM binding site conforms to a "1-10" CaM binding motif, whereas the second motif conforms to a "1-5-8-14" CaM binding motif (Fig 1D). These motifs are named for the placement of bulky hydrophobic residues within a CaM target sequence [34,36]. Additionally, 1-5-8-14 motifs also tend to carry a net positive charge of +3 or greater. Unlike IQ motifs that classically bind CaM in the absence of Ca 2+ , these two motifs typically bind CaM only in the presence of Ca 2+ .
This region of PC1 is well conserved among PC1 orthologs, with higher conservation of the second putative CaM binding motif than the first motif ( Fig 1C). The second predicted CaMbinding domain also overlaps with the previously identified G-protein activation domain [7], highlighting the importance of this region to the function of PC1.

The PC1 tail interacts with Ca 2+ /CaM
The data from these in silico analyses led us to hypothesize that the PC1 tail may interact with calmodulin in the presence of calcium. To experimentally test whether PC1 binds CaM, membrane-anchored constructs containing the full-length PC1 tail (FLM), or the N-terminal (NTM), or C-terminal (CTM) halves of the PC1 tail (Fig 2A) were expressed in HEK293T cells and incubated with CaM agarose beads. As shown in Fig 2B, the full-length and N-terminal half of the PC1 tail bind strongly to CaM, whereas the C-terminal half does not. This is consistent with the location of the predicted CaM binding sites within the N-terminal half of the PC1 tail.
To test whether the binding between PC1 and CaM is dependent on Ca 2+ , similar CaM-precipitation experiments with the PC1 tail were carried out either in the absence or presence of Ca 2+ (200 μM buffered Ca 2+ ). As shown in Fig 2C, CaM binding to PC1 depends on Ca 2+ as predicted.
CaM has four EF-hands that each bind to Ca 2+ with different affinities. To further investigate the dependency of the PC1/CaM interaction on Ca 2+ , binding experiments were carried out with a range of free Ca 2+ concentrations (Fig 2D and 2E). The results demonstrate that PC1-FLM binds to CaM at very low concentrations of Ca 2+ , suggesting that only the high affinity EF-hands of CaM need to be bound to Ca 2+ for the interaction to occur.
Next, point mutations were introduced in the PC1 tail in an attempt to disrupt the interaction with CaM. Mutagenesis of the first predicted CaM binding motif alone, without altering ). The two outlined myosin sequences identify the two IQ motifs that bind myosin light chains (ELC or RLC). (B) A structural model of the PC1 tail based on the known structure of scallop myosin. (C) Alignment of the human PC1 sequence with known orthologs (Mus musculus, NP_038658; Canis lupus familiaris, NP_001006651; Gallus gallus, XP_015149930; Xenopus tropicalis, XP_012826626; Takifugu rubripes, XP_011610747). The two IQ-like motifs are boxed in red, the previously identified G-protein activation sequence is underlined, the second predicted CaM binding motif, did not disrupt the overall ability of the PC1 tail to bind CaM (Fig 2F). In contrast, mutating the well-conserved serine (S4144) in the second domain to an aspartic acid resulted in strong attenuation of binding between CaM and the PC1 tail ( Fig 2G). By sequence analysis [35], the S4144D mutation is predicted to eliminate CaM binding in the second putative CaM binding motif, presumably by reducing the basic charge that is characteristic of the 1-5-8-14 motifs (Fig 1C).
Furthermore, deletion of the first putative CaM binding motif from the PC1 tail retained binding to CaM, but this binding was largely eliminated by simultaneous mutation of serine 4144 ( Fig 1H). Finally, we also found that a previously identified patient mutation that occurs within the second putative CaM-binding motif (L4137P) strongly attenuated CaM binding ( Fig 2I). This mutation was previously classified as "Highly Likely Pathogenic" [37].
The PC1 constructs used in binding experiments for Fig 2G-2I are non-membraneanchored, soluble versions of the PC1 tail that mimic the native cleavage product of PC1 termed PC1-p30. We and others have shown that PC1-p30 is overexpressed in polycystic kidneys, targets to the nucleus and regulates the activities of several transcription factors including STAT3, STAT6, TCF and CHOP [10-12, 17, 27]. Therefore, these results suggest that PC1 can interact with CaM irrespective of its proteolytic processing and membrane-anchorage.
Altogether, these results identify the second predicted CaM binding motif as the major CaM binding site in the cytoplasmic tail of PC1. The interaction is Ca 2+ -dependent, and mutation of this motif results in overall reduced CaM binding to PC1 that may be associated with disease pathogenesis. These findings led us to hypothesize that CaM binding to PC1 may be necessary for one or more PC1-mediated functions.
Mutation of the CaM binding motif does not affect PC1's ciliary localization nor its regulation of AP1 and STAT3 signaling Full-length, membrane-anchored PC1 localizes to the endoplasmic reticulum [20,38] and primary cilia [5] of renal epithelial cells [9][10][11]13]. To investigate how CaM binding may affect the function or localization of PC1, we generated stable MDCK renal epithelial cell lines that express either wild-type PC1 or PC1 carrying the S->D mutation that disrupts the main CaM binding site. For these experiments, mouse PC1 was used instead of human PC1 because the available mouse expression construct results in much more robust expression. The sequences of the cytoplasmic tails are extremely conserved between mouse and human and the equivalent mutation (S4134D) was introduced to disrupt CaM binding. Expression of PC1 in these cell lines can be controlled due to the use of a doxycycline-inducible promoter.
In fully polarized MDCK cells grown on Transwell filters, PC1 largely localized to primary cilia ( Fig 3A). PC1-S4134D also localizes strongly to primary cilia, suggesting that CaM binding is not necessary for the cilia targeting of PC1. This finding was confirmed by incubating the cells with the CaM inhibitor W13 which did not affect the cilia localization of PC1 (Fig 3B).
PC1 and PC2 are known to interact via coiled-coil domains in their cytoplasmic tails [24][25][26]39]. Although the CaM binding motif in PC1 is not very near the coiled-coil domain, we tested whether mutation of the CaM binding motif affects the PC1-PC2 interaction. Wild-type or S4134D-mutant full-length PC1 were co-expressed with PC2 in HEK293T cells and their and the mutated serine that results in attenuated CaM binding is identified with an asterisk. (D) Alignment of the two putative CaM binding domains in PC1 with proteins containing known CaM binding motifs (NP_002212.3, NP_851276, NP_001160158, NP_002882.3, NP_000611.1, NP_001093422.2, NP_014626, NP_476895, NP_001027.3). Hydrophobic amino acids at characteristic positions within each sequence are highlighted. The first predicted CaM binding domain in PC1 (above) best resembles a 1-10 motif, with bulky hydrophobic residues at positions 1 and 10 in the sequence. The second predicted domain (below) resembles a 1-5-8-14 motif, with bulky hydrophobic residues at these positions and a characteristic +3 charge.  interaction analyzed by co-immunoprecipitation. Both wild-type and mutant PC1 bound equally well to PC2 (Fig 3C), suggesting that CaM binding is not necessary for the PC1-PC2 interaction.
PC1 has been reported to activate AP1/JNK signaling via activation of heterotrimeric Gproteins [9]. Importantly the region of the PC1 tail that activates heterotrimeric G-proteins [7] overlaps significantly with the CaM binding motif identified here ( Fig 1C). Therefore we tested the effect of the CaM-binding mutant in an AP1 luciferase reporter assay. As shown in Fig 4A, both the wild-type and mutant constructs of the PC1 tail activate AP1 signaling equally well, suggesting that CaM binding is not necessary for PC1-mediated G-protein signaling and AP1/ JNK activation.
We have previously shown that the membrane-bound PC1 tail can activate STAT3 via the tyrosine kinase JAK2 [10]. Additionally we identified patient mutations that mapped near the CaM binding site that affected this activity [10]. To test whether STAT3 activation is dependent on CaM binding, a STAT3 luciferase reporter assay was used. Both the wild-type membrane-anchored PC1 tail and the CaM-binding mutant led to similar STAT3 activation ( Fig  4B) indicating that CaM binding is not required for STAT3 activation by membrane bound PC1.
We also reported that the soluble, cleaved PC1 tail (PC1-p30) can co-activate STAT3 after IL6 activation [10]. To test whether this function of PC1 is dependent on CaM binding we tested PC1-p30 and the corresponding CaM-binding mutant using a STAT3 reporter assay in cells that were stimulated with IL6 ( Fig 4C). Again, both wild-type and mutant PC1-p30 coactivated STAT3 similarly indicating that this function is not dependent on CaM binding.
Additionally, we previously reported that the tyrosine kinase Src can bind PC1-p30 and activate STAT3 in a JAK2-independent manner [11]. Therefore we tested our CaM binding mutant in this functional assay as well but, again, wild-type and mutant PC1-p30 led to similar Src-dependent STAT3 activation ( Fig 4D). Consistent with this, the CaM inhibitor W13 had no effect in this assay ( Fig 4E).
Together, these results indicate that CaM binding to the PC1 C-terminal tail is not required for cilia targeting or the ability of PC1 to signal via AP1 or STAT3.
To identify other signaling pathways that may be regulated by CaM binding to PC1, we analyzed lysates from the DOX-inducible cells expressing either wild-type or CaM-binding mutant PC1 (S2 Fig). We found that expression of wild-type PC1 reduces phospho-S6 levels consistent with the previous finding that PC1 suppresses mTORC1 signaling [15,40,41]. In contrast, the mutant PC1 does not affect phospho-S6 levels suggesting that CaM binding is required for mTOR regulation by PC1. Phospho-Erk and phospho-STAT3 levels, on the other hand, do not change with PC1 expression (S2 Fig). These data suggest that CaM binding to PC1 may only regulate specific PC1-dependent signaling pathways.

PC1/PC2-mediated channel activity is disrupted by CaM inhibition
PC1 is thought to function in a complex with PC2 as a mechano-sensitive Ca 2+ -permeable ion channel in the primary cilium [5,42]. Thus, the inducible PC1-expressing MDCK cell lines were used to test the potential role of CaM-binding in flow-dependent Ca 2+ signaling. Similar  Regulation of Polycystin-1 Function by Calmodulin Binding to previous reports [5] polarized MDCK cells produce a transient rise in cytosolic Ca 2+ in response to apical fluid flow (Fig 5A). Upon doxycycline-induction to over-express wild-type PC1, MDCK cells exhibit a significantly larger Ca 2+ increase in response to flow (Fig 5A and  5B). In contrast, MDCK cells expressing the CaM-binding mutant of PC1 exhibit a strongly suppressed Ca 2+ increase in response to flow. This suggests that the mutant form of PC1 acts as a dominant negative inhibitor with regard to flow-induced Ca 2+ signaling, presumably by displacing endogenous, wild-type PC1 in these cells. Given that the CaM-binding mutant of PC1 still efficiently targets to cilia ( Fig 3A) and still binds to PC2 (Fig 3C) this suggests that the PC1/PC2 channel complex depends on CaM binding to mediate flow-dependent signaling.
In order to understand the dependence of this effect on flow rate, we measured calcium levels for each cell line at 0, 0.5, 1, and 8 dyne/ cm 2 (S3 Fig). MDCK cells responded optimally to a flow rate of 1 dyne/ cm 2 . However calcium levels were elevated in wild-type PC1-expressing cells and suppressed in mutant PC1-expressing cells across all flow rates tested.
To verify that the effect is indeed due to CaM inhibition, the response of PC1-expressing MDCK cells to flow was tested in the presence of the CaM inhibitors W13 vs. the control compound W12. These inhibitors were used at levels well below their established IC 50 concentrations to avoid any potential off-target effects. Despite this, we still noticed a significant decrease in flow-induced Ca 2+ signaling in cells treated with W13, but not with the control compound W12 (Fig 5C and 5D). Together these data suggest that CaM binding to the PC1 tail regulates flow-induced Ca 2+ signaling.
To investigate the channel activity of the PC1/PC2 complex more directly we measured La 3+ -sensitive whole cell macroscopic currents from CHO cells transiently expressing both Regulation of Polycystin-1 Function by Calmodulin Binding PC1 and GFP-tagged PC2 as described previously [43]. CHO cells expressing both proteins produced strong currents that were eliminated in the presence of La 3+ (Fig 6A). PC1 containing the CaM-binding mutation produced significantly weaker currents than wild-type PC1 (Fig 6B  and 6C), suggesting that PC1's interaction with CaM is required for full PC1/PC2 channel activity.
Altogether these results indicate that PC1-dependent channel activity is enhanced by binding of CaM to the motif identified here, and that inhibition of this interaction reduces channel activity and flow-dependent Ca 2+ signaling.
Expression of the CaM-binding mutant of PC1 alters cellular respiration PC1 has recently been shown to regulate cell metabolism [41]. To test the potential role of CaM binding to PC1 on cell metabolism we measured the oxygen consumption rates (OCR) of DOX-inducible MDCK cells expressing wild-type or mutant PC1 using a Seahorse cell metabolism analyzer in the absence of fluid flow. Cells induced to express wild-type PC1 exhibited slightly higher, albeit not significant (p = 0.08), basal mitochondrial respiration (Fig 7A and  7C) and ATP-linked respiration (Fig 7D and S4 Fig) relative to uninduced controls. In contrast, Regulation of Polycystin-1 Function by Calmodulin Binding cells expressing the CaM-binding mutant exhibited sharply lower oxygen consumption rates (Fig 7B and S4 Fig), reflecting lower basal mitochondrial respiration (Fig 7C), ATP-linked respiration (Fig 7D), and maximal mitochondrial respiration (Fig 7E).
These data support a previous report [41] that PC1 is an important regulator of cell metabolism and further suggest that CaM binding to PC1 is necessary for the normal regulation of this function. As measurements were performed in the absence of apical fluid flow, these metabolic changes are likely independent of cilia-dependent flow-sensing.

Discussion
Here we report for the first time that the PC1 C-terminal tail has potential structural homology to the regulatory domain of myosin heavy chains and contains two predicted CaM-binding motifs. We confirm the ability of CaM to interact with the second predicted CaM-binding motif, and identify a mutant that largely disrupts this interaction.
The sequence similarity we identified between regions of PC1 and myosin led us to hypothesize that these regions may have similar interaction partners. However, it remains unclear what the evolutionary relationship is between these two proteins. As they share relatively little sequence homology outside of the identified region, it is possible that the similarity is due to convergent evolution.
PC1 is not the only ciliopathy-related protein that interacts with CaM. Nephrocystin 5 and inversin both have two IQ domains, interact with CaM, and localize to cilia [44][45][46]. It was also found that CaM itself localizes to cilia [44], suggesting that this is where these proteins interact. Given that bending of primary cilia results in increased cytosolic Ca 2+ [5], CaM binding may be a general strategy to regulate signaling by ciliary proteins, and thus mutations in these regulatory regions may explain some causes of disease.
We have previously shown that a PEST motif within the C-terminal tail of PC1 regulates its protein stability [12]. Furthermore this motif has recently been shown to be necessary for calpain-mediated degradation of PC1 [47]. CaM binding domains are commonly associated with PEST sequences, however the full functional implications of this association remains unclear [48]. It is tempting to speculate that CaM binding to PC1 may regulate the stability and signaling of the polycystin complex by regulating access to the PEST domain.
We found that binding of CaM to the identified binding motif in PC1 is not required for PC1's function in promoting STAT3 or AP1 signaling (Fig 4). This was surprising given that this region of PC1 also interacts with JAK2 [10] and heterotrimeric G-proteins [7]. We found, however, that inhibition of CaM binding results in decreased PC1/PC2 Ca 2+ channel activity and potently inhibits flow-dependent Ca 2+ signaling. These results suggest that CaM serves to regulate the PC1/PC2 ion channel complex. The polycystins have been found by several investigators to localize both to primary cilia and the ER. It is possible that CaM binding to PC1 in the ER may regulate different Ca 2+ -sensitive processes than it does in the primary cilium. Previously reported ER-associated functions of PC1 that may be regulated by CaM binding include regulation of store-operated Ca 2+ entry (SOCE) [20,49] and IP 3 receptor-dependent Ca 2+ release [38,50].
We and others have also described a PC1-p30 cleavage product that translocates to the nucleus and regulates gene transcription. Although calmodulin still binds this fragment, we were unable to identify a function for this interaction. Given the accumulation of this fragment in ADPKD tissue, it remains an important question for future investigation.
We found that expression of the CaM-binding mutant of PC1 alters cellular metabolism. This is consistent with previous reports that have shown that PC1 plays a role in regulating cellular metabolism [41,60]. The exact mechanism through which this occurs remains poorly understood. However, our results suggest that this function requires CaM binding and likely full PC1/PC2 channel activity.
Our data are consistent with a model in which calmodulin binds PC1 in response to elevated calcium. This in turn leads to full activation of the PC1-PC2 ion channel complex, either in the cilium or elsewhere in the cell (ie the ER), which may be necessary to regulate mitochondrialdependent metabolism. It seems likely that activation of the PC1-PC2 complex is not only a response to calmodulin binding but is also the cause, by raising local calcium concentrations sufficiently to initiate calmodulin binding. However it is also possible that other sources of calcium trigger binding of PC1 to calmodulin.
Altogether, our results add CaM as a new player to the multitude of binding partners of PC1. Given that CaM binding to PC1 is Ca 2+ dependent, this could provide an important mechanism for regulating the functions of the polycystins.

Plasmids
The constructs for PC1-FLM, PC1-CTM, PC1-NTM, and PC1-p30 (aka PC1-FLS) have been previously described [12]. All of the other constructs of the PC1 tail were made by PCR-based Quikchange site-directed mutagenesis (Agilent Technologies, cat# 200521) from these original constructs. PC1-p30ΔC1 is a soluble construct made from a deletion of PC1-p30 and encodes amino acids Y4127-T4303 of PC1. All plasmids are made in pcDNA4/TO (Invitrogen), and empty vector was used as a negative control in calmodulin binding experiments. A PKD2 construct with an amino-terminal GFP tag was generated by the ligation of a PCR fragment that included the start codon and open reading frame of GFP in the 5'-multiple cloning region of a previously described PKD2 expression vector [24], followed by site directed mutagenesis resulting in an in-frame fusion transcript. Mutant and wild-type full-length mouse PKD1 expression constructs in a pcDNA4 backbone were made by first excising the majority of the 5'-region of PKD1 from the pcDNA3 expression construct kindly provided Stefan Somlo [61] using unique XbaI and XhoI sites and replacing it with filler DNA. The remaining 3'-PKD1 gene region was transferred into a modified pcDNA4/TO/2xMyc-6xHis (Invitrogen) backbone using the resulting unique XbaI and AgeI sites. The 2xMyc-6xHis tag was moved in frame as well as mutations in S4144 using site directed mutagenesis. The final full-length product was then reassembled using the XbaI and XhoI products from the original construct.

Cell culture
HEK293T cells were acquired from ATCC and grown in DMEM (Corning Cellgro) + 10% FBS (Omega Scientific) + 1X Penicillin/ Streptomycin (Corning Cellgro) at 37°C in 5% CO 2 . Stable MDCK cells containing the tetracycline repressor (pcDNA6/TR) have been previously described [62]. To make inducible full-length PC1 expressing cells, we transfected these cells using the calcium phosphate method. Stable cells were selected using 300 μg/mL Zeocin and clones were selected based on their doxycycline-induced expression of the transgene. MDCK cell lines were cultured in MEM + 5% FBS + 1X Penicillin/ Streptomycin at 37°C in 5% CO2. Unless otherwise noted, expression was induced for 16 hours using 50ng/L of doxycycline diluted in growth media.

Calmodulin binding experiments
For transient expression, HEK293T cells were plated on 6-well plates at a confluence of approximately 70% and incubated overnight to promote adherence. The next day transfection complex was made in 250 μL Optimem (Life Technologies) that contained 5 μL of Turbofect (Thermo Scientific) and 2μg of DNA per condition. Transfection complex was added to the medium and incubated with cells for an additional 16 hours.
Lysates were made from HEK293T cells expressing PC1-tail constructs by lysing them with 200 μL lysis buffer (50 mM Tris-HCl pH7.4, 100 mM KCl, 1% Triton X-100, 5 mM MgCl 2 + protease inhibitors) containing the desired concentration of free calcium. Non-zero free calcium concentrations made by using 5 mM CaCl 2 and varying the amount of EGTA were determined using the MaxChelator tool (http://www.stanford.edu/~cpatton/maxc.html). Conditions lacking calcium contained 5 mM EGTA only. Lysates were cleared by centrifugation at 13,000 rpm for 10 minutes. Supernatants were tumbled at 4°C with 30 μL of prewashed CaM-agarose beads for 1 hour. Precipitates were washed 3 times in lysis buffer and analyzed by immuno-blot.

Luciferase reporter assays
The AP1 and STAT3 reporter assays were performed similarly. HEK293T cells were plated onto 12-well plates and transfected with 250 ng luciferase reporter, 20 ng β-galactosidase, and 250 ng of the gene of interest. The gene of interest for control samples was EGFP in pcDNA4. Transfection complexes were formed by combining the DNA with 2 μL Lipofectamine 2000 (Life Technologies) in 250 μL Optimem (Life Technologies). Complexes were added to cells and incubated for 4 hours. After incubation the media were changed for complete media. 25 ng/mL IL6 was added as indicated. Cells were incubated for another 16 hours lysed in 100 uL passive lysis buffer (Promega, Cat# E1941) for 30 minutes shaking at room temperature. Lysate was analyzed for luciferase activity using the Luciferase Assay System (Promega, Cat# 1501) and β-galactosidase activity. β-galactosidase activity was measured by adding 10 μL of lysate to ONPG buffer (100 mM NaPhosphate pH7.4, 1 mg/mL ONPG, 130 mM 2-mercaptoethanol, 3 mM MgCl 2 ) and reading the absorbance at A405 when the samples turned light yellow. Luciferase activity values were normalized by β-galactosidase activity. Control conditions were assigned a value of one and all other values were compared to the control. For Fig 4D and 4E, STAT3 activity of PC1-p30/ Src-expressing cells was set to 100, and all other measurements were normalized to this.

Alignments and PC1 structural modeling
Pairwise sequence alignments were performed between the sequence of the PC1 tail and proteins of known structure. A portion of the PC1 tail (4100-4158) was found to have~25% identity and 30% similarity to the sequence of the heavy chain of the scallop myosin regulatory domain 777-836 (PDB_ID: 1SCM). Based on this alignment an approximate 3D model of the corresponding PKD1 fragment was made using programs COOT [63] and CHAIN, a modified version of FRODO [64]. The model has retained the myosin α helical fold. The N-terminal helical part of the model became slightly longer because an Ala insertion at 4112. The single amino acid deletions corresponding to a Leu and Gly in the myosin sequence were regularized with restrain to the α helical motif. Orientations of the side chains were chosen to avoid steric conflicts.

Immunofluorescence microscopy
MDCK cells were grown on Transwell polycarbonate membranes (Corning) for 8-10 days with growth media containing serum below the filter and growth media lacking serum above the filter. Expression of PC1 was induced 16-hours prior to fixation with 50ng/L of doxycycline. Cells were fixed in -20°C methanol for 15 minutes. The methanol was rinsed 2X 2 minutes in TBS. The cells were incubated in cell block and permeabilization buffer (CBP; 2% BSA, 0.2% Triton-X100, 0.05% sodium azide in TBS) for 1 hour at 37°C. Transwells were then cut out and incubated with primary antibodies diluted in CBP overnight at 4°C. The next day they were washed 3X 5 minutes in wash buffer (0.7% fish skin gelatin, 0.05% triton X-100, in TBS) then incubated with fluorescent-conjugated secondary antibodies for 1 hour at 37°C. The Transwells were again washed 3X 5 minutes in wash buffer, followed by two rinses with TBS. The antibodies were fixed to their antigens by post-fixation for 10 minutes in 10% neutral-buffered formalin. They were then washed 2X 5 minutes in TBS and mounted onto a slide and coverslipped using Prolong Gold mounting media with DAPI (Molecular Probes). Images were taken with a Fluoview 1000 confocal laser scanning microscope. Image stacks were processed and analyzed using Image J.

Measurement of flow-dependent calcium transients
After grown to confluence, stable MDCK cell lines were treated with either 1 μL PBS or doxycyline (final concentration 50 ng/L) diluted in 10 mL media for about 36 hours. Cytosolic calcium was measured using a Nikon TiE microscope, and cells were challenged with fluid-shear stress of about 1 dyne/cm 2 as previously described [66]. Briefly, cells were loaded for 30 minutes at 37°C with 5 mM Fura-2 AM (Invitrogen, Inc.). After being washed to remove excess Fura-2 AM, cells were placed and observed under the microscope for optimal fluorescence signals.
After equilibrated for 10 minutes, cells were challenged at fluid-shear stress of 0, 0.5, 0.8, 1.0 dyne/cm 2 . Pairs of intracellular calcium images were captured every five seconds at the excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm. These images were captured through Fura-2 filter kit that contains 25mm 340/380 excitation filters, a dichroic mirror, and a wide band 510 nm emission filter. For better focusing, the microscope was equipped with XY-axis motorized flat top inverted stage and Nikon automatic focusing RFA Z-axis drive. For a better controlled environment, the body of the microscope was enclosed inside a custom built chamber to control CO 2 , humidity, heat and light.

Measurement of oxygen consumption rate (OCR) of cells by Seahorse XF24 analyzer
An XF24 analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used to measure the bioenergetic function of the MDCK (PC1) or MDCK (PC1-S4134D) cells. Briefly, cells were seeded at a density of 30,000 cells per well and grown in the presence or absence of 50 ng/L doxycycline. On the day of the assay, medium was replaced by unbuffered DMEM supplemented with 1 g/L glucose. OCR was measured before and after the sequential injection of 1 μM oligomycin (ATP synthase inhibitor), 0.25 μM FCCP (mitochondrial uncoupler), and 0.75 μM of rotenone (complex 1 inhibitor). Mix, wait, and measure times were 3, 2, and 3 minutes, respectively. The mitochondrial function parameters determined were: 1) basal respiration (difference between OCR of untreated cells and cells incubated with rotenone), 2) ATPlinked respiration (difference between OCR of untreated cells and cells treated with oligomycin), 3) maximal respiration (difference between OCR of cells treated with FCCP and cells with rotenone treatment).