Sds22, a PP1 phosphatase regulatory subunit, regulates epithelial cell polarity and shape [Sds22 in epithelial morphology]

<p>Abstract</p> <p>Background</p> <p>How epithelial cells adopt their particular polarised forms is poorly understood. In a screen for genes regulating epithelial morphology in <it>Drosophila</it>, we identified <it>sds22</it>, a conserved gene previously characterised in yeast.</p> <p>Results</p> <p>In the columnar epithelia of imaginal discs or follicle cells, mutation of <it>sds22 </it>causes contraction of cells along their apical-basal axis, resulting in a more cuboidal morphology. In addition, the mutant cells can also display altered cell polarity, forming multiple layers in follicle cells and leaving the epithelium in imaginal discs. In yeast, <it>sds22 </it>encodes a PP1 phosphatase regulatory subunit. Consistent with this, we show that <it>Drosophila </it>Sds22 binds to all four <it>Drosophila </it>PP1s and shares an overlapping phenotype with <it>PP1beta9c</it>. We also show that two previously postulated PP1 targets, Spaghetti Squash and Moesin are hyper-phosphorylated in <it>sds22 </it>mutants. This function is shared by the human homologue of Sds22, PPP1R7.</p> <p>Conclusion</p> <p>Sds22 is a conserved PP1 phosphatase regulatory subunit that controls cell shape and polarity.</p

Control of Tissue Growth and Cell Transformation by the Salvador/Warts/Hippo Pathway

The Salvador-Warts-Hippo (SWH) pathway is an important regulator of tissue growth that is frequently subverted in human cancer. The key oncoprotein of the SWH pathway is the transcriptional co-activator, Yes-associated protein (YAP). YAP promotes tissue growth and transformation of cultured cells by interacting with transcriptional regulatory proteins via its WW domains, or, in the case of the TEAD1-4 transcription factors, an N-terminal binding domain. YAP possesses a putative transactivation domain in its C-terminus that is necessary to stimulate transcription factors in vitro, but its requirement for YAP function has not been investigated in detail. Interestingly, whilst the WW domains and TEAD-binding domain are highly conserved in the Drosophila melanogaster YAP orthologue, Yorkie, the majority of the C-terminal region of YAP is not present in Yorkie. To investigate this apparent conundrum, we assessed the functional roles of the YAP and Yorkie C-termini. We found that these regions were not required for Yorkie's ability to drive tissue growth in vivo, or YAP's ability to promote anchorage-independent growth or resistance to contact inhibition. However, the YAP transactivation domain was required for YAP's ability to induce cell migration and invasion. Moreover, a role for the YAP transactivation domain in cell transformation was uncovered when the YAP WW domains were mutated together with the transactivation domain. This shows that YAP can promote cell transformation in a flexible manner, presumably by contacting transcriptional regulatory proteins either via its WW domains or its transactivation domain

YAP's transactivation domain is dispensable for its ability to stimulate cell proliferation.

<p>(a) Proliferation rate of NIH-3T3 cells expressing vector alone (CON) or the indicated YAP plasmids when cultured in medium containing 0.5% serum. (b) Quantitation of the number of colonies expressing vector alone (CON) or the indicated YAP plasmids in MCF10A cells grown in soft agar for 14 days. Data in (a) and (b) is presented as mean +/− SD, n = 3. (c) Representative pictures of acini of MCF10A cells quantified in (b).</p

YAP does not require its transactivation domain to transform cells.

<p>(a and b) Quantitation of the number of colonies expressing vector alone (CON) or the indicated YAP plasmids in MCF10A (a) or NIH-3T3 (b) cells grown in soft agar. (c) Representative pictures of soft agar assays from (b). (d) Quantitation of the size of colonies expressing the indicated plasmids in NIH-3T3 cells grown in soft agar. Data in a, b and d are presented as mean +/− SD, n = 3.</p

The carboxyl terminus of Yorkie is dispensable for its ability to stimulate tissue growth in <i>D. melanogaster</i>.

<p>(a–c) Dorsal views of fly heads expressing the indicated transgenes with the eye-specific <i>GMR-Gal4</i> driver. (d–f) Wings of flies expressing the indicated transgenes using the <i>71B-Gal4</i> driver. (g) Quantification of wing sizes of genotypes displayed in (d–f). Data is presented as mean +/− SD, n = 20 for each genotype, *** indicates p<0.0001. (h and i) Expression of <i>Yki</i> (h) and <i>Yki-ΔC</i> (i) in the posterior compartment of the developing wing (marked by GFP, green) with the <i>en-Gal4</i> driver resulted in upregulation of <i>ex-lacZ</i> (grayscale in single channel, red in overlay). (j–l) <i>yki^B5</i> mutant clones alone or co-expressing a <i>yki</i> transgene in wing discs, marked by GFP (green). Nuclei of cells are marked with DAPI (blue). (j) <i>yki^B5</i> mutant clones. (k) <i>yki^B5</i> mutant clones co-expressing <i>Yki</i>. (l) <i>yki^B5</i> mutant clones co-expressing <i>YkiΔC</i>.</p

Hyperactivated YAP requires WW domains and TEAD transcription factors to stimulate cell transformation.

<p>Quantitation of number of colonies expressing vector alone (CON) or the indicated YAP plasmids in MCF10A cells (a) or NIH-3T3 cells (b). Data is presented as mean +/− SD, n = 3.</p

YAP requires its transactivation domain to promote cell migration and invasion.

<p>(a) Quantitation of acini with invasive protrusions in MCF10A cells expressing vector alone (CON) or the indicated YAP plasmids. (b) Quantitation of enclosed area after a scratch was introduced for 20 hours in confluent MCF10A cells expressing vector alone (CON) or the indicated YAP plasmids. Data are presented as mean +/− SD, n = 3.</p

Schematic illustration of wild-type and mutant Yorkie and YAP proteins.

<p>Wild-type Yki is 418 amino acids long, whereas Yki-ΔC lacks the final 51 amino acids at the C-terminus. YAP2L is 504 amino acids long and contains two WW domains, as well as three domains in its C-terminus: an SH3 binding domain, a transactivation domain (TA) and a PDZ-binding motif. In YAP-ΔC, the C-terminus of YAP is deleted. In YAP-ΔTA, the TA domain is deleted. These deletions were generated in wild-type YAP2L, as well as in YAP2L-S127A, which contains a single amino acid mutation of S127 to A. In YAP-S127A-ΔTA-S94A, S94 is also mutated to A. YAP-WW1+2* includes W199F and P202A mutations in WW domain 1 and W258F and P261A mutations in WW domain 2. In YAP-WW1+2*-ΔTA, the WW domains are mutated as above and the TA domain is deleted.</p