Phosphorylation of Yun is required for stem cell proliferation and tumorigenesis

Abstract Stem cells maintain adult tissue homeostasis under physiological conditions. Uncontrolled stem cell proliferation will lead to tumorigenesis. How stem cell proliferation is precisely controlled is still not fully understood. Phosphorylation of Yun is essential for ISC proliferation. Yun is essential for the proliferation of normal and transformed intestinal stem cells. Our mass spectrometry and biochemical data suggest that Yun can be phosphorylated at multiple residues in vivo. Interestingly, we show that the phosphorylation among these residues is likely interdependent. Furthermore, phosphorylation of each residue in Yun is important for its function in ISC proliferation regulation. Thus, our study unveils the important role of post‐translational modification of Yun in stem cell proliferation.


| INTRODUCTION
Tissue homeostasis is maintained by residential stem cells, which proliferate and produce differentiated progeny to replenish lost cells. Thus, the proliferation (self-renewal) and differentiation of adult stem cells must be tightly balanced under physiological conditions. Disruption of this balance will result in excessive stem cell or precocious stem cell differentiation and finally stem cell depletion, eventually leading to various diseases, such as cancer and precocious aging. [1][2][3] In particular, it has been proposed that various tumours possess cancer stem cells (CSCs), which are the driving force of tumour development and progression, dormancy, and recurrence. [4][5][6] Therefore, illustrating the underlying mechanisms of stem cell proliferation control is critical for understanding homeostasis regulation and for the development of potential therapeutics to treat human diseases including cancer.
The adult Drosophila intestine is an excellent model to study the regulation of stem cell proliferation/differentiation and tumorigenesis, which shares marked similarities in terms of development, cellular makeup, and genetic control with its mammalian counterparts. [7][8][9][10][11] Drosophila intestinal stem cells (ISCs) are distributed along the basement membrane of the adult intestinal epithelium 12,13 and divided asymmetrically to produce differentiating enteroblasts (EBs) or EE progenitors (EEPs). One of the Notch ligands, Delta, is specifically expressed in ISCs, while the Notch receptor is expressed in ISCs, EBs, and EEPs (termed progenitors collectively). Notch signalling activation in EBs promotes their differentiation into absorptive enterocytes (ECs). 12,[14][15][16][17] Recent studies show that EE cells may not be generated from EBs, but directly from ISCs or EEPs, which divide once to produce two EEs. [18][19][20] The proliferation and differentiation of ISCs are regulated by multiple signalling pathways such as the Notch, Wingless (Wg), JAK/STAT, EGFR, Hippo, Insulin, Hedgehog, and BMP signalling to maintain tissue homeostasis under physiological and stressed conditions (see reviews [21][22][23][24] and references therein).
In our recent study, we identified a novel intrinsic factor Yun (Luck in Chinese) which sustains the proliferation of normal and transformed ISCs. 25 Yun is a novel protein without any known domains or motifs.
During the course of our study, another group showed that it is implicated in cell proliferation in larval brain and spermatogenesis with unknown mechanisms and named it as diamond (dind). 26 Yun is required for the proliferation of ISCs under physiological conditions and tissue regeneration under stress conditions. 25 The identification of Yun as an essential stem cell proliferation regulator has important applications, which can be used as a target to treat intestinal malignancies. However, it remains unexplored whether its activity in ISC proliferation is regulated by any post-translational modifications (PTMs).
In this study, we investigate whether PTMs exist in Yun and whether these PTMs are required for Yun's function in ISC proliferation. We find that Yun is phosphorylated in vivo. Several phosphorylation residues are identified by mass spectrometry. Site-directed mutagenesis analyses indicate that the phosphorylation of these residues may be interdependent and is important for the proper function of Yun in ISC proliferation regulation.

| Yun is a positive regulator of ISC proliferation
To identify regulators involved in ISC maintenance and proliferation/ differentiation, we carried out a genome-wide RNAi screen in Drosophila adult posterior midgut using an esgGal4, UAS-GFP, tubGal80 ts (esg ts ) driver, which is expressed in the progenitors (ISCs, EBs, and EEPs). 25,27,28 From the screen, we identified many known and novel ISC regulators, including Yun (encoded by CG7705). 25 Yun encodes a novel protein without any known domains or motifs. We examined its expression pattern by immunostaining using a Yun-specific antibody and found that in adult intestines, Yun was mainly expressed in progenitors and EEs ( Figure S1).  Figure S4).

| Yun is required for tumorigenesis
We then explored whether yun is also required for tumorigenesis.   L). Collectively, these data demonstrate that Yun is essential for tumorigenesis (and CSC proliferation).

| Yun is post-translationally modified by phosphorylation in vivo
To explore the mechanism of how Yun regulates ISC proliferation and tumorigenesis, we immunoprecipitated endogenous Yun proteins with a 3XFlag tag at its C-terminus under the control of its endogenous promoter. 25   (E) Quantification of pYun/total Yun ratio of different Yun forms. Mean ± SD is shown. n = 3. ***p < 0.001; ****p < 0.0001 Legend on next page.

| Phosphorylation of different residues of Yun may be interdependent
To further confirm that Yun protein is phosphorylated in vivo, we pulled down Yun by IP experiments and detected it with phosphorylated serine and/or threonine (pS/T)-and phosphorylated serine (pS)specific antibodies, respectively. The results showed that Yun could be detected by both antibodies, supporting the notion that Yun is phosphorylated at serine residue(s) in vivo ( Figure 4A). Consistently, phosphorylation of Yun could also be detected when analysed with phos-tag ( Figure 4B). Next, we treated the immunoprecipitated Yun proteins with λ-phosphatase (λ-PP), which could de-phosphorylate the phosphorylated proteins. The results showed that treatment with λ-PP could effectively diminish phosphorylation of Yun proteins ( Figure 4B,C). Together, these data show that Yun protein is posttranslationally modified by phosphorylation in vivo.
We further examined the contribution of phosphorylation status of Yun by these candidate phosphorylation residues and whether the phosphorylation status of individual residue can affect phosphorylation of the other residues. We performed site-directed mutagenesis of these residues, changing each of them to alanine to make phosphorylation dead mutants, and generated transgenic flies carrying these mutants individually ( Figure 4D). We then examined the effects of these single sites on the phosphorylation status of Yun using phostag. We found that mutation of each single serine residue significantly affected, but did not completely abolish the phosphorylation of Yun protein, supporting the notion that Yun protein is phosphorylated at multiple serine residues ( Figure 4D). Of note, mutation of each single serine residue affected the phosphorylation status of Yun at different extents, implying that phosphorylation of individual residue may affect the phosphorylation status of the other residue(s), thus the phosphorylation of these residues may be interdependent ( Figure 4D,E).

| Phosphorylation of Yun is critical for ISC proliferation
Protein phosphorylation plays an important roles in regulating protein activity and function. 32,33 To collectively address the role of phosphorylation of these serine residues in ISC proliferation regulation, we  (Figures 5N-T). Collectively, these data suggest that phosphorylation of all the five serine residues identified is important for Yun to regulate ISC proliferation, but the role(s) played by individual serine residue may be different and phosphorylation statuses among these residues are likely interdependent.

| DISCUSSION
The proliferation and differentiation of adult stem cells must be tightly balanced in order to maintain tissue homeostasis and prevent tumori- Hpo. [36][37][38][39][40][41] The two casein kinases are essential for ISC proliferation.
depletion of them compleletly blocks ISC proliferation. Importantly, ectopic expression of both wildtype yun and phosphorylation mimic yun(5SD) could not restore the proliferation defects in the absence of either casein kinase, suggesting that multiple downstream substrates are required for ISC proliferation control and expression of single substrate is not sufficient to restore ISC proliferation blockage upon their depletion ( Figure S5).
Our identification of Yun as a phosphorylated protein provides an interesting cutting point to elucidate the underlying mechanism of how Yun regulates ISC proliferation and tumorigenesis. It will be interesting to systematically investigate how these serine residues of Yun are differentially phosphorylated in ISCs, whether any interdependence(s) of phosphorylation status exists among these residues, and how the phosphorylation of these residues is coordinated to regulate the function of

| RNAi knockdown and overexpression experiments
Crosses (unless stated otherwise) were maintained at 18 C to bypass potential requirements during early developmental stages. Twothree-days-old progeny with the desired genotypes were collected after eclosion and maintained at 29 C to inactivate Gal80 ts before dissection and immunostaining. The flies were transferred to new vials with fresh food every day.

| MARCM clone analysis
The clonal analyses were achieved using the MARCM system. 29 The ISC clones were induced by heat shocking Two-three-days-old adult flies at 37 C for 60 min. The flies were maintained at a 25 C incubator and transferred to new vials with fresh food every day. The sizes of the marked clones were assayed at 6 days after clone induction (6D ACI, at least 10 midguts for each genotype were assayed).

| Database search
The MS data were searched against a Uniprot Drosophila melanogaster protein database (database ID number of UP000000803) using ProLuCID with the following parameters: precursor mass tolerance, 3 Da; fragment mass tolerance 20 ppm; peptide length, minimum 6 amino acids and maximum 100 amino acids; enzyme, Trypsin, with up to three missed cleavage sites. 43 The results were filtered by DTASelect requiring FDR <1% at the peptide level and spectra count≥2. 44 The proteins identified from the negative control and Flag-Yun IP were contrasted by Contrast. 44

| Data analysis
The number of intestines scored is indicated in the text. To determine the relative number of esg + cells, confocal images of 40Â lens/1.0 zoom from a defined posterior midgut region of different genotypes indicated were acquired. The number of esg + cells from each confocal image was determined using Image-Pro Plus software, manually selecting the "filter" depending on the respective cell size to filter out background signals (referred to as the relative number of esg + cells).
The clone sizes were scored manually under Zeiss Imager Z2/LSM780 microscope for indicated genotypes. At least 10 different guts were analysed for each set. Statistical analysis was done using the Student's t-test. PEMS 3.1 software was used for SEM analyses and Sigma plot and GraphPad prism software for graph generation. The graphs were further modified using Adobe Photoshop and Illustrator. ns p >0.05; *p < 0.05; **p < 0.01; *** p < 0.001; and **** p < 0.0001.