Does Calcineurin Regulate the Osmotic Stress Response in S

Does Calcineurin Regulate the Osmotic Stress Response in S. cerevisiae Through the Smp1 Transcription Factor?

Danni Lu May, 2016

Does Calcineurin Regulate the Osmotic Stress Response in S. cerevisiae Through the Smp1 Transcription Factor?

An Honors Thesis Submitted to the Department of Biology in partial fulfillment of the Honors Program STANFORD UNIVERSITY

by Danni Lu May, 2016

Table of Contents ______

Title Page...... 2 Signature Page...... 3 Table of Contents...... 4 Abstract...... 5 Introduction...... 7 Materials and Methods...... 13 Yeast strains, media, and growth...... 13 E. coli transformations...... 13 Purification of His-tagged calcineurin for in vitro assays...... 14 GST fusion protein expression for pulldown binding assay...... 14 Immunoblotting...... 15 Yeast transformations and cloning...... 15 Plasmids...... 16 STL1-LacZ assay...... 16 Results...... 17 Does full-length GST-Smp1 bind to yeast calcineurin in vitro?...... 17 Does calcineurin interact with Smp1 at a PxIxIT or PxIxIT/D-site?...... 17 Does calcineurin act during the Hog1 MAPK response to osmotic stress?...... 18 Does calcineurin regulate Hog1-dependent gene expression?...... 20 Discussion...... 22 Tables...... 26 Figures...... 28 References...... 37

4 Abstract

Calcineurin is a conserved Ca2+/calmodulin-regulated phosphatase that regulates the T cell immune response, as well as many other processes, and is the direct target of immunosuppressant drugs (cyclosporin A and FK506). Because of its ubiquitous expression and prevalence in a broad spectrum of disorders, long term suppression of calcineurin also results in harmful side effects (Safa, Riella, and Chandraker 2013). The lack of sequence similarity in bona fide calcineurin substrates, save for conserved calcineurin docking sites ('PxIxIT' and 'LxVP' ), has hindered candidate substrate identification. However, there have been recent efforts in systematically screening potential substrates for these docking sites. This study investigates the possible interaction between calcineurin and Smp1, a yeast transcription factor that is activated via phosphorylation by Hog1 MAP Kinase (Hog1 MAPK) under hyperosmotic conditions.

Using computational analysis, all possible 6-mers in Smp1 were screened for the potential to interact with calcineurin as a ‘PxIxIT’ site, generating seven high-scoring peptide sequences. The goal of this project was to investigate the potential interaction between Smp1 and calcineurin, using two approaches: 1) testing for direct binding of

Smp1 and calcineurin in vitro, and 2) studying regulation of Smp1 activity by calcineurin in the presence of osmotic stress in vivo. Co-purification assays revealed that full length

GST-tagged Smp1 binds to yeast calcineurin in vitro, and may bind at one of the computationally predicted ‘PxIxIT’ sites. In vivo functional assays revealed that calcineurin both positively and negatively regulated Smp1 activity, suggesting that calcineurin may regulate the Hog1 MAPK pathway through multiple substrates. Overall, these results suggest that calcineurin is intimately involved in the Hog1 MAPK pathway,

5 and substantiates this computational approach for de novo identification of calcineurin interaction sites. This approach may be used for future identification of novel calcineurin substrates, and provide insight on how to selectively target calcineurin substrates.

6 Introduction

Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine phosphatase that, in mammals, regulates T-cell activation through the NFAT (nuclear factor of activated T-cells) family of transcription factors. Calcineurin also regulates neuronal differentiation and activity, cardiac development and hypertrophy, as well as skeletal muscle fiber-type specification. Cyclosporin A and FK506 are both drugs that directly target and block calcineurin, and are used in humans for organ transplantation to suppress the immune system and prevent tissue rejection (Safa, Riella, and Chandraker

2013). However, given calcineurin's ubiquity in many signaling processes, inhibition of this phosphatase using immunosuppressant drugs can result in undesired side effects. In

Saccharomyces cerevisiae (baker's yeast), calcineurin is necessary for survival in several different types of environmental stress (Cyert 2003). There have recently been efforts to screen the yeast proteome systematically to identify calcineurin substrates (Goldman et al. 2014). The discovery of new calcineurin substrates would increase our current understanding of the mechanism of calcineurin-substrate interaction and help to further elucidate the calcineurin signaling network. This will allow the development of new techniques to more specifically target certain calcineurin substrates.

Structure of calcineurin

Calcineurin is a heterodimeric molecule that consists of a catalytic A subunit

(CNA) and a regulatory B subunit (CNB). It also possesses an autoinhibitory domain

(AID) on the C-terminus of CNA that interacts with the catalytic site and prevents dephosphorylation of calcineurin substrates under basal cytosolic conditions (Fig. 1). In

7 the presence of elevated Ca2+ ion levels, calmodulin binds to the calmodulin-binding domain of CNA and displaces the AID, allowing the catalytic site to interact and dephosphorylate substrates. Ca2+ ions will also bind directly to the CNB subunit, causing a conformational change that helps to release the AID and further exposes the active site

(Aramburu, Rao, and Klee 2000).

Calcineurin substrate recognition through the PxIxIT site

Phosphatase substrates contain little sequence similarity, given that phosphatases have a limited preference for amino acids flanking the dephosphorylated residues. It is known, however, that calcineurin contains a conserved ß1-4 sheet on the CNA subunit that recognizes the short linear motif (SLiM) ‘PxIxIT’ present in substrate proteins and aids in the docking of the substrate onto calcineurin (Grigoriu et al. 2013). The PxIxIT motif refers to the consensus sequence found in calcineurin substrates:

[P][ˆPG][IVLF][ˆPG][IVLF][TSHEDQNKR], where positions 2 and 4 exclude the amino acids following the '^' symbol. Different SLiMs containing this sequence have been expressed and are found to dock successfully onto calcineurin. This includes PVIVIT, a high-affinity binder to the calcineurin PxIxIT docking site (Li et al. 2007).

Experiments have shown that altering the PxIxIT sequence on the well studied calcineurin substrate, Crz1 (a known yeast substrate of calcineurin), significantly affects the binding-affinity of calcineurin to Crz1. Crz1 is a transcription factor regulated by calcineurin in a similar manner that the NFAT transcription factors are regulated by calcineurin in humans. Mutations in the Crz1 PxIxIT site that change it to a higher or lower affinity site correspondingly result in higher or lower affinity binding of Crz1 to

8 calcineurin and higher or lower amounts of calcineurin regulated Crz1 activity (Roy et al.

2007). This study demonstrates how the PxIxIT docking site mediates calcineurin- substrate interaction and can determine calcineurin substrate activity.

Searching for new calcineurin substrates

In order to fully elucidate the calcineurin-substrate network, Goldman et al. developed a systematic approach to identifying calcineurin substrates. Using mass spectrometry and bioinformatics, a large number of candidate substrates were identified that contained a potential PxIxIT site and whose phosphorylation was regulated by calcineurin. These candidates were further screened using co-purification and dephosphorylation assays to establish bona fide calcineurin substrates and/or interacting partners in yeast (Goldman et al. 2014), validating the identification of PxIxIT sites as a method to find calcineurin substrates.

Interestingly, some substrates appeared to exhibit overlapping calcineurin/MAPK docking sites ('PxIxIT/D-sites'), suggesting that some kinases may directly compete with calcineurin in determining the phosphorylation state of these substrates (Fig. 2). This

PxIxIT/D-site is characterized by a PxIxIT sequence preceded by three to five basic

amino acid residues, and can be notated as (Ψ)3-5PxΦxΦζ (Φ, hydrophobic residue; ζ, hydrophilic residue; Ψ, basic residue), One such example was found in the protein Dig2, which is a target of the Fus3 mitogen-activated protein kinase (MAPK) pheromone response pathway (Goldman et al. 2014). Several other proteins in the yeast genome contained sequences that matched this combined PxIxIT/D motif, and were thus candidates to be competitively regulated by MAPK and calcineurin. One of these proteins

9 is the yeast transcription factor Smp1 which is activated under osmotic stress conditions by the Hog1 MAPK (De Nadal et al. 2003).

Other substrates identified in the screen included synaptojanin (Inp52/Sjl2), and the closely related Inp53/Sjl3, which are activated by calcineurin under conditions of osmotic stress. Osmotic stress has been shown to cause a rise in cytosolic Ca2+ in yeast, through the release of Ca2+ by the vacuolar membrane protein, Yvc1p (Denis 2002). This allows calcineurin to be activated and to localize to actin patches, where it regulates

Inp53, and repolarizes the cell. Inp53 also appears to activate endocytic proteins, which helps to regulate the cell membrane and adapt to a decreased turgor pressure caused by a hypertonic solution (Guiney et al. 2015).

This provides the basis for the investigation of the yeast proteome for proteins that mainly play a role under conditions of osmotic stress. The Goldman et al. study conducted a screening for all yeast proteins that were hyper-phosphorylated in calcineurin-deficient extract under wild-type conditions; thus, proteins that are only phosphorylated under osmotic stress, such as Smp1, would not be expected to be identified in that study. This motivated us to directly test if Smp1 was a calcineurin substrate.

Smp1 and the osmotic stress response

Smp1 is a member of the MEF2C family of transcription factors, a family that has been reportedly targeted by mammalian p38 MAPK in metazoan cells. MEF2C transcription factors are involved in a number of processes, including early cardiogenesis through the regulation of angiopoietin 1 and VEGF, as well as craniofacial development

10 (Bi, Drake, and Schwarz 1999; Verzi et al. 2007). Smp1 is one of several yeast transcription factors that are phosphorylated under osmotic stress by the Hog1 mitogen- activated protein kinase (Hog1 MAPK) (Fig. 3). Smp1 then activates the expression of genes such as STL1, which encodes a glycerol proton transporter, as an osmoadaptation response (De Nadal et al. 2003). Given that calcineurin has been shown to be active in conditions of osmotic stress and that Smp1 is phosphorylated by Hog1 MAPK, we postulated that Smp1 may be a potential calcineurin substrate, and that calcineurin may directly compete with Hog1 for recognition of Smp1, thus down-regulating the Hog1

MAPK osmotic stress response pathway.

Using yeast as a model organism to study calcineurin-substrate interaction

Calcineurin is highly conserved between yeast and mammals. Given the ease of genetic and biochemical manipulation, S. cerevisiae serves as an ideal model organism to study calcineurin and its interaction with potential substrates. It has a rapid growth rate and simple nutritional requirements, making it easier to rigorously control experiment conditions using selective dropout media. Of particular interest is the ability to introduce foreign genes into cells using plasmid vector systems. By transforming DNA into yeast, mutant strains can be generated that can produce a target protein in high copy (Stearns,

Ma, and Botstein 1990). This greatly expands the versatility of this species as model organisms for studying proteins-protein interaction through genetic and biochemical manipulation.

11 A multifaceted approach to studying calcineurin and Smp1 interaction

My project utilizes the DNA transformation techniques described in the above section to introduce into E. coli full-length Smp1 or short peptides derived from the protein fused with a GST tag. These fusion proteins were then tested for binding to calcineurin in vitro. Western blot analysis was used to image and interpret the results.

In addition, a computational approach was used to determine if Smp1 contained an overlapping PxIxIT/D site or any other PxIxIT-like calcineurin docking sites (Nikhil

Damle, unpublished results). Seven different high-scoring 6-mers were fused to GST to create plasmids pDL1-7, and tested to see if they bind calcineurin. In each case, 15 amino acids encompassing the 6-mers and flanking amino acids from Smp1 were N-terminally fused to GST, transformed into E. coli, expressed in high copy, and then collected in extract to be tested for interaction with calcineurin in vitro, in a similar method as the full-length GST-Smp1.

My project also utilizes a transcriptional reporter gene in several yeast strains

(wild-type, hog1Δ, and smp1Δ) to directly measure the Hog1 MAPK response pathway during osmotic stress in vivo. My project tested the effect of calcineurin on the wild-type yeast strain by adding FK506, the calcineurin inhibitor, to cells grown in the presence of

NaCl or CaCl2 stress. A plasmid that expresses a constitutively active form of calcineurin was also expressed in this strain and tested under the same osmotic stress conditions.

Both the in vivo and in vitro results provide evidence that calcineurin interacts with Smp1 through a complex manner, and plays an important role in the Hog1 MAPK osmotic stress response in yeast.

12 Materials and Methods

Yeast strains, media, and growth

The yeast strains used in this project were from the Cyert laboratory stock as well as provided by the Posas lab. A list of strains many be found in Table 1. Yeast was grown at 30°C, unless specified. The yeast media was either Yeast Peptone Dextrose (YPD) or

Synthetically Defined (SD). Media was made following procedures from Sherman

(Sherman 2002), but with the following changes: the amount of each amino acid included was doubled and NH4Cl (3.5%) was used in place of (NH4)2SO4.

E. coli transformations

E. coli were transformed using the electroporation or heat shock method. Ligated oligos were first inserted into DH10ß electrocompetent E. coli cells DNA using the electroporation method. Approximately 3µL of the ligated oligos were added to ~100µL

DH10ß cells, then pulsed with a BTXTM Personal Electroporation Pak, followed immediately addition of 500µL Luria Broth (LB) media for recovery. After one hour at

37°C, cells were plated on LB + Ampicillin selective plates. DNA from these cells was purified using QIAGEN kits as per the manufacturer's protocols.

DNA was transformed into E. coli strain BL21-DE3 using the heat shock method.

1µL of DNA was added to a 50µL aliquot of BL21-DE3 cells, and kept on ice for 30 minutes. Following this period, the cells were heat shocked at 37°C for exactly 10 seconds, and then immediately put on ice. The cells were then grown in 950µL Super

Optimal Broth (SOC) at room temperature for 1 hour, followed by plating on selective media, LB + Ampicillin.

13 Purification of His-tagged calcineurin for in vitro assays

Constructs were created for wild-type such that the Cna1 can be expressed as a N- terminal 6-HIS fusion from an IPTG inducible promoter in bacteria. BL21 E. coli cells were co-transformed with CNA1-p11 plasmid and pET-CNB1 plasmid expressing Cnb1.

500 mL transformants were grown to mid-log phase (OD600 = 0.8) and induced with

1mM IPTG for 16 hours at 18°C . Cells were pelleted, washed and pellets frozen at -

80°C. Cell pellets were subsequently resuspended in lysis buffer (50 mM Tris-HCl pH 8,

150 mM NaCl, 0.1% Triton X-100 1mM BME and protease inhibitors) and lysed by sonication. Unlysed cells were cleared by centrifugation. The cell lysate was centrifuged twice at 13,000rpm for 20 minutes and the supernatant was loaded on a Ni-NTA-agarose column. The column was washed in wash buffer (50mM Tris-HCl, 5mM imidazole,

500mM NaCl, pH 8 at 4C) and the proteins eluted in 200µL fractions in elution buffer

(50mM Tris-HCl, 500mM imidazole, 500mM NaCl, pH 8 at 4°C). Eluted proteins were checked by Bradford assay for protein concentration and by gel electrophoresis and staining with gel-code blue. Appropriate fractions were pooled and dialyzed in 50mM

Tris-pH 7.5 and 150mM NaCl. Dialyzed proteins were stored at -80°C.

GST fusion protein expression for pulldown binding assay

Expression of GST fusion proteins was carried out using the protocol outlined in

Rodriguez and Roy (Rodríguez et al. 2010). E. coli strain BL21 was transformed with bacterial plasmids containing the GST fusion proteins. Addition of 1mM isopropyl-D- thiogalactopyranoside (IPTG) induced synthesis of the GST fusion proteins for 16 hours

14 at 18°C while shaking. Protein extracts were stored at -80 °C. Protein concentrations were measured using Bradford assays.

Immunoblotting

The procedure followed that outlined in Roy et al. (Roy et al. 2007). Equivalent amounts of protein were loaded into an acrylamide gel and then transferred to nitrocellulose paper. The nitrocellulose blot was blocked overnight in a 10% milk-TBST

(Tris base, saline and tween buffer) solution and then probed for 1 hour with an anti-CNA antibody and an anti-GST antibody. The antibodies were washed off with TBST and a blot was probed with the appropriate secondary antibody for 1 hour. The secondary antibody was washed off and the blot was then visualized using a LiCOR scanner.

Yeast transformations and cloning

Yeast cells were transformed using the lithium acetate method. 50mL of yeast strain YEN2 at OD600 = 0.15-0.2 was grown until log phase, or OD600 = 0.6-0.9. Cells were then spun down at 3000rpm for 7 minutes, and washed with 10mL 1X TE (0.1M

Tris-HCl pH 7.5, 0.01M EDTA). Cells were spun to pellet as before, now, then washed with 10mL 1X LiAc (lithium acetate, pH 7.5)/1X TE mix, then spun to pellet as before.

Pellet was resuspended in 500µL 1X LiAc/1X TE, and aliquots of 75-100µL were made per transformation tube. The appropriate DNA and 20µg carrier DNA was then added to each transformation tube, followed by 700µL PEG mix, immediately mixing. Tubes were then incubated at 30°C for 30 minutes on an end-over-end rotator, followed by heat shock in a 42°C water bath for 15 minutes, and pelleted at 6000rpm for 2 minutes. Supernatant

15 was aspirated, and washed with 0.5mL dH2O, and washed and aspirated as before.

Finally, each tube was resuspended in 0.25mL dH2O, and plated on selective media.

Plasmids

A list of plasmids used may be found in Table 2. The generation of several of the plasmids required cloning, and the cloning procedure used may be obtained upon request from the Cyert lab.

STL1-LacZ transcriptional activity assay

This assay followed the procedure as outlined in Stathopoulos et al.(Stathopoulos and

Cyert 1997) Cells grown to saturation were diluted to an OD600 of 0.2 and were then grown to mid-log phase (~0.6-0.8 OD). During this time, if needed, the calcineurin inhibitor FK506 or vehicle ethanol-tween (ET) was added to a final concentration of

10µg/mL. Once in mid-log phase, the cells were induced under varying concentrations of

NaCl or CaCl2 for 35 minutes. Cells were then pelleted and lysed to obtain protein extracts by the following procedure outlined in Withee et al (Withee et al. 1997). Protein concentrations were measured using the Bio-Rad Bradford Assay to ensure that equivalent amounts of extract were being compared. A Benchmark Plus Bio-Rad spectrophotometer was used to measure the intensity of the yellow color produced by the breakdown of ONPG (O-nitrophenyl-ß-D-galactopyranoside, obtained from Sigma) by ß- galactosidase. The intensity of the yellow color, which is the result of the breakdown product o-nitrophenol, was used to determine the level of ß-galactosidase activity.

16 Results

Does full-length GST-Smp1 binds to yeast calcineurin in vitro?

We first wanted to ascertain if Smp1 and calcineurin directly interacted with each other. In vivo experiments were unsuccessful due to complications in expressing Smp1, and so an in vitro approach was taken. Equal amounts of purified His-tagged yeast calcineurin were pulled down by Ni-NTA-agarose beads in the presence of bacterial cell lysate that contained a plasmid expressing full length GST-tagged Smp1 (Fig. 4). GST-

Dig2 (a confirmed calcineurin substrate) and GST (vector) served as the positive and negative controls, respectively.

The co-purification of GST-Smp1 with calcineurin in Fig. 4 confirmed the direct interaction with calcineurin in vitro. Increasing the amount of cell lysate from 100µg to

200µg of total protein while keeping calcineurin amounts constant correlated with an increased amount of GST-Smp1 that co-precipitated.

These results show that GST-Smp1 directly interacts with calcineurin in vitro.

Does calcineurin interact with Smp1 at a PxIxIT or PxIxIT/D-site?

We next wanted to determine if this interaction between Smp1 and calcineurin was mediated by a PxIxIT site or a 'PxIxIT/D-site. For this hypothesis, a computational approach was developed based on the 3-D structure of PxIxIT peptides binding to calcineurin (Damle et al., unpublished). This approach uses the binding energies of six- amino acid peptides to the PxIxIT docking groove on calcineurin as well as several other secondary structure criteria. Scores were assigned based on values for amino acid interaction preferences determined by Jha et al. (Jha, Vishveshwara, and Banavar 2010).

17 A total of seven high-scoring ~15-amino acid SLiMs (pDL1-7) were generated, each N-terminally fused to GST (Table 3), including two candidate PxIxIT/D-sites, pDL1 and pDL6. These SLiMs were then tested for in vitro binding as above (Fig. 5).

GST (vector) served as the negative control, while GST-Dig2 or GST-VIVIT served as the positive control. GST-VIVIT is a short peptide sequence that binds with high affinity to the PxIxIT-docking site on calcineurin. Fig. 5A shows that pDL1-3 show little to no binding to calcineurin. Fig. 5B shows that pDL4 and pDL5 appear to bind to calcineurin, while pDL6 does not. Finally, Fig. 5C shows that pDL7 also shows little to no binding to calcineurin.

These binding assays initially suggested that pDL4 and pDL5 both co- precipitated with calcineurin. We further wanted to ensure that this co-purification was specific to calcineurin. Thus we included an additional control experiment, where co- purification was performed in the presence or absence of calcineurin to check for non- specific binding (Fig. 6). This revealed that pDL4 bound non-specifically to Ni-NTA agarose in the absence of calcineurin. Note that the two candidate PxIxIT/D-sites, pDL1 and pDL6, were tested and found not to bind to calcineurin in vitro.

These experiments suggest that calcineurin interacts with Smp1 directly and revealed one SLiM, pDL5, to be a potential PxIxIT site on Smp1 that mediates the interaction.

Does calcineurin act during the Hog1 MAPK response to osmotic stress?

We next wanted to examine if calcineurin regulates Smp1 activity in vivo. The

Hog1 MAPK pathway is activated by the addition of NaCl to the media in a growing

18 culture of yeast cells. Since calcineurin is best activated with extracellular calcium added to the media (Roy et.al. 2007), we also used extracellular calcium to activate both calcineurin and the Hog1 MAPK pathways. Therefore, we first tested whether the Hog1

MAPK pathway could be activated in the presence of 0.2M CaCl2, which should also create a hyperosmotic stress condition.

To test whether 0.2M CaCl2 activated the Hog1 MAPK osmotic stress response pathway, several strains of yeast (wild-type, hog1Δ, and smp1Δ), each containing a STL1-

LacZ reporter gene (an reporter for Smp1 transcriptional activity), were subjected to both

NaCl and CaCl2 osmotic stress in equivalent amounts (Fig. 7) (De Nadal et al. 2003).

Similar to the results seen in De Nadal et al. (De Nadal et al. 2003), for the wild- type strain, there was an approximately 3 fold increase in STL1-LacZ activity with the addition of NaCl, compared to the basal level (no addition). This confirms that STL1-

LacZ activity is activated by osmotic stress as previously observed. The addition of CaCl2 resulted in a similar increase. Thus like NaCl, CaCl2 produces osmotic stress, which is

Hog1 dependent, as the response is not observed in the hog1Δ strain. In smp1Δ, we observed a reduced osmotic stress response for both NaCl and CaCl2 stimulation, establishing, as observed previously, that Smp1 and other transcription factors activate

STL1 expression.

The results of this experiment show that the osmotic stress response in the presence of CaCl2, like NaCl, is completely dependent on Hog1 MAPK, and partially dependent on Smp1. This is consistent with results showing that the Hog1 MAPK phosphorylates a number of transcription factors, including Smp1, to regulate the

19 expression of STL1 (De Nadal et al. 2003). This confirms that CaCl2 can be used to activate both calcineurin and Hog1 MAPK.

Does calcineurin regulate Hog1-dependent gene expression?

Given that Hog1 MAPK activates Smp1 by phosphorylation, we now wanted to investigate whether calcineurin may dephosphorylate Smp1 and therefore down-regulate the osmotic stress response. We tested this hypothesis by inhibiting calcineurin with

FK506 in the presence of CaCl2 stress in wild-type yeast cells (Fig. 8). If calcineurin antagonizes the Hog1 MAPK pathway, then inhibiting calcineurin would result in an elevated osmotic stress response. Compared to the sample with ET (vehicle), the addition of FK506 more than doubled the osmotic stress response in CaCl2. Interestingly, this same effect was not seen after the addition of NaCl, where calcineurin is not expected to be activated to the same degree.

Conversely, we also wanted to test the effect of calcineurin on the Hog1 MAPK pathway when calcineurin is constitutively activated. If calcineurin antagonizes the Hog1

MAPK response, we hypothesized that the presence of constitutively active calcineurin would suppress the response to osmotic stress. We introduced a plasmid expressing constitutively active calcineurin (CNtrunc) into wild-type cells, and tested for the osmotic stress response in the absence or presence of CaCl2 (Fig. 9). This form of calcineurin is missing the auto-inhibitory domain from the C-terminus of the CNA subunit, leaving the active site exposed. When cells were stimulated with a low amount of CaCl2 (50 mM) we did not observe any change in STL1-LacZ activity between control cells and cells expressing CNtrunc. This indicates that constitutively active calcineurin is having little

20 effect under these conditions. However, when we further activated calcineurin with increased CaCl2 (100 mM), cells containing CNtrunc showed almost double the osmotic stress response. This is contrary to our original hypothesis, and suggests that calcineurin may be activating the Hog1 pathway under these conditions. These conflicting results suggest that calcineurin plays a complex role in the Hog1 MAPK pathway that goes beyond dephosphorylating and inactivating Smp1.

21 Discussion

The aim of this project was to determine whether calcineurin directly interacted with the yeast transcription factor, Smp1, and regulated its activity in the Hog1 MAPK osmotic stress response pathway. We studied the possible interaction between calcineurin and Smp1 in vitro and found that it may bind to calcineurin at a possible PxIxIT-like sequence on Smp1, 231SNYHNFYPSPYEN243. Interestingly, this sequence is not contained in either the MADS box or MEF2 domains in Smp1, nor in the C-terminal

Hog1 MAPK-interacting region of Smp1 (De Nadal et al. 2003). Thus, this region may be available for interaction with calcineurin. Note that it is not one of the original predicted PxIxIT/D-sites that were originally identified. This sequence also does not exactly conform to the consensus PxIxIT motif that is typically observed. For example, the first position is an ‘F,’ which is an interesting deviation form the consensus sequence.

In order to test for direct protein-protein interaction between Smp1 and calcineurin, we used pull-down binding assays to test whether Smp1 would interact with purified calcineurin. We also expressed several additional candidate PxIxIT-like sequences from Smp1, and initially found that two (pDL4 and pDL5) co-purified with calcineurin.

After conducting a follow-up experiment with a bait-free (calcineurin-free) control, we found that pDL4 co-purified with the Ni-NTA beads, suggesting that this

SLiM bound non-specifically to the beads. Future experiments should be conducted to confirm that pDL5 is not binding non-specifically to the beads.

If GST-Smp1 does indeed bind specifically to calcineurin via the pDL5 site, then a number of tests could be conducted to determine whether or not pDL5 acts as a PxIxIT

22 docking site. One test would be to mutate the essential amino acids in the PxIxIT consensus of pDL5 (FYPSPY → AYASAA) and assess binding of this sequence to calcineurin. Mutating these amino acids to alanines would reduce binding to calcineurin if it were a PxIxIT site. These same mutations could then be made in the full-length

Smp1 as well and tested for binding to calcineurin.

Another method to test for PxIxIT-like binding would be to conduct competition binding assays of pDL5 with the PVIVIT peptide, which is a high-affinity binder to calcineurin (Li et al. 2007). Increasing the presence of the PVIVIT peptide in the in vitro binding assays would reduce the number of exposed sites on calcineurin for PxIxIT sites to dock. Therefore, less pDL5 would be pulled down when compared to the same assay without PVIVIT peptide being present. This would confirm that pDL5 indeed acts like a

PxIxIT docking site. Similar binding assays would ideally be performed in vivo to determine if calcineurin interacts with Smp1 within a cell environment.

When we examined the effect of calcineurin on Smp1 function in vivo, we found seemingly contradictory results, based on our original hypothesis. In the case of the basic model that our hypothesis suggests, calcineurin directly dephosphorylates Smp1, which would suppress the activity of Smp1 in the presence of osmotic stress. This would directly antagonize the activity of Hog1 MAPK, which phosphorylates Smp1.

Therefore, we expected that inhibition of endogenously expressed calcineurin would relieve any suppression of Smp1 transcriptional activity under osmotic stress, which was supported by our results. Inversely, expressing constitutively active CNtrunc would up-regulate suppression of Smp1 and the osmotic stress response. However, expressing CNtrunc resulted in increased STL1-LacZ activity, which indicates an increased

23 osmotic stress response. One possible explanation that may reconcile these results may be that calcineurin also regulates other transcription factors involved in the Hog1 MAPK pathway. Hog1 MAPK is known to phosphorylate Sko1, Hot1, and the redundant Msn2 and Msn4 (De Nadal et al. 2003). Note that STL1, which we used as the reporter gene, is a downstream product of the Hog1 MAPK pathway, and its presence may be affected by several of these transcription factors. Even if calcineurin did, in fact, suppress the transcriptional activity of Smp1 (which would reduce STL1-LacZ activity), calcineurin may be regulating these other transcription factors such that CNtrunc may cause an overall net increase in osmotic stress response, masking the effect of calcineurin on Smp1.

Calcineurin may also be acting on a protein upstream of Smp1. Since STL1 is a downstream product in the Hog1 MAPK pathway, another method would have to be used to directly test for Smp1 activity, such as measuring phosphorylation levels of Smp1 or directly testing if calcineurin can dephosphorylate Smp1 in vitro.

Furthermore, Bai et al. have recently confirmed that Hot1, one of the transcription factors under the regulation of Hog1 MAPK, is critical in activating STL1 (Bai, Tesker, and Engelberg 2015). It may be worthwhile to perform similar experiments that were conducted in this project with Smp1 on Hot1, since STL1 would be a more reporter of the effect of calcineurin on Hot1.

In conclusion, the in vitro binding assays suggest that calcineurin may bind to

Smp1 at a potential PxIxIT site, pDL5. The in vivo activity assays show that calcineurin regulates the Hog1 MAPK osmotic stress response in a complex process that may involve several substrates within this pathway. Confirmation of the interaction between calcineurin and Smp1 in the future would substantiate the computational approach our lab

24 has taken to identify potential PxIxIT sites in candidate substrates. This would allow us to broaden its use to the human proteome and create a human calcineurin signaling network.

Understanding how such calcineurin substrates are interconnected may allow us to more specifically target certain proteins and develop new immunosuppressant drugs.

Tables

Table 1: Yeast Strains Used in This Study Strain Genotype Source

YEN2 MAT a, leu2, trp1, his3, STL1-LacZ (de Nadal et al., 2002) URA3

YEN7 MAT α, leu2, trp1, his3, lys2, hog1 (de Nadal et al., 2002) ::TRP1 STL1-LacZ URA3

MAT a, leu2, trp1, his3 smp1 ::HIS3 YEN48 STL1-LacZ URA3 (de Nadal et al., 2002)

Table 2: Plasmids Used in This Study Plasmid Description Source pRS314 Empty vector This study pJR82 CNA1trunc-pRS314 This study

Figures

Figure 1: Calcineurin molecule interacting with A238L, a protein inhibitor from African swine fever virus, known to inhibit calcineurin and NFkB. The surface representation of CNA (gray) and CNB (beige) are pictured with the CNA active site highlighted in cyan. The PxIxIT (PKIIIT) and LxVP (LCVK) sites of the viral peptide interacts with calcineurin in the PxIxIT and LxVP binding pockets (yellow and green, respectively) (Grigoriu et. al 2013).

Figure 2: Overlapping PxIxIT/D-site in Dig2. Consensus residues in PxIxIT (∗) and D- site (underlined) are shown. Φ, hydrophobic residue; ζ, hydrophilic residue; Ψ, basic residue (Goldman et al. 2014)

Figure 3: The HOG1 MAPK signaling pathway. Hog1 is activated by Pbs2 after being exposed to osmotic stress, causing it to localize to the nucleus and phosphorylate a number of transcription factors, including Smp1(Hersen et al. 2008).

Figure 4: Full-length Smp1 coprecipitates with yeast calcineurin (CN). Strain BL21 E. coli cells were transformed with a plasmid expressing GST-Smp1, lysed, and the extract was collected. Dig2, GST, and yeast calcineurin were also expressed using the same procedure, followed by column and dialysis purification. Samples were taken before and after being affinity purified using Ni-NTA beads, with calcineurin as the bait protein, and the cell lysates (containing 100µg, 200µg, or 500µg of total protein) assessed for co- elution by Western blot.

Figure 5: pDL5 coprecipitates with yeast calcineurin. Strain BL21 E. coli cells were transformed with a plasmid expressing pDL1-7 fused with a GST-tag, lysed, and the extract was collected. Dig2, GST, GST-VIVIT, and yeast calcineurin (CN) were also expressed using the same procedure, followed by column and dialysis purification. Samples were taken before and after being affinity purified using Ni-NTA beads, with calcineurin as the bait protein, and the GST proteins assessed for co-elution by Western blot.

Figure 6: pDL4 non-specifically binds to yeast calcineurin. Strain BL21 E. coli cells were transformed with a plasmid expressing pDL1-7 fused with a GST-tag, lysed, and the extract was collected. Dig2, GST, GST-VIVIT, and yeast calcineurin (CN) were also expressed using the same procedure, followed by column and dialysis purification. Samples were taken before and after being affinity purified using Ni-NTA beads, with or without calcineurin as the bait protein, and the GST proteins assessed for co-elution by Western blot.

STL1-LacZ Ac.vity

600

500

400

300 None

NaCl 200 CaCl2 ß-galactosidase ac.vity

100

0 Wild-type hog1Δ smp1Δ

Yeast Strain

Figure 7: Hog1 MAPK osmotic stress response is reduced in hog1Δ and smp1Δ strains. Yeast cells were grown in YPD to mid-log phase, then not subjected (black bars) or subjected to osmotic stress (0.4M NaCl = gray bars, 0.2M CaCl2 = white bars). The cells were lysed following this stress period, and the cell extracts were assayed for ß- galactosidase activity in the cell, using STL1-LacZ as a downstream reporter. ß- galactosidase activity was measured by the addition of ONPG to the extracts, using a spectrophotometer to measure relative levels of o-nitrophenol, a yellow breakdown product.

STL1-LacZ Ac.vity (Strain: wild-type)

1200

1000

800

None 600 NaCl

CaCl2 400

ß-galactosidase ac.vity 200

0 ET (vehicle) FK506

Figure 8: STL1-LacZ expression is induced under CaCl2 osmotic stress in the presence of FK506. Wild-type cells containing the STL1-LacZ reporter system (strain YEN2) were grown to mid-log phase, were subjected to addition of ET (vehicle) or FK506 for one hour, and then were subjected to or not subjected to hyperosmotic stress (no osmotic stress, 0.4M NaCl for 35 mins, or 0.2M CaCl2 for 35 mins). The cells were lysed following this stress period, and the cell extracts were assayed for ß-galactosidase activity in the cell. ß-galactosidase activity was measured by the addition of ONPG to the extracts, using a spectrophotometer to measure relative levels of o-nitrophenol, a yellow breakdown product.

STL1-LacZ Ac.vity (Strain: wild-type + CNtrunc)

1200

1000

800

Control 600 CNtrunc 400

ß-galactosidase ac.vity 200

0 50mM CaCl2 100mM CaCl2

Figure 9: STL1-LacZ expression is induced under CaCl2 osmotic stress in the presence of constitutively active calcineurin. Wild-type cells containing the STL1-LacZ reporter system (strain YEN2) were transformed with a control plasmid or a multi-copy plasmid expressing a constitutively active truncated calcineurin. These cells were grown to mid- log phase and then were subjected to CaCl2 hyperosmotic stress (0.05M, 0.10M CaCl2 for 35 mins). The cells were lysed following this stress period, and the cell extracts were assayed for ß-galactosidase activity in the cell. ß-galactosidase activity was measured by the addition of ONPG to the extracts, using a spectrophotometer to measure relative levels of o-nitrophenol, a yellow breakdown product.

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