Phosphorylation of Grainy head by ERK is essential for wound-dependent regeneration but not for development of an epidermal barrier Myungjin Kima and William McGinnisa,1 aSection of Cell and Developmental Biology, University of California at San Diego, La Jolla, CA 92093-0349 Edited* by Michael Levine, University of California, Berkeley, CA, and approved December 9, 2010 (received for review November 2, 2010) Grainy head (GRH) is a key transcription factor responsible for that cross-link the specialized apical extracellular matrix mole- epidermal barrier formation and repair, whose function is highly cules that make up the cuticle (4, 6). GRH also is required for conserved across diverse animal species. However, it is not known normal expression levels of Fasciclin 3 and Coracle proteins, how GRH function is reactivated to repair differentiated epidermal which are directly involved in mediating epidermal cell adhesion barriers after wounding. Here, we show that GRH is directly re- (4, 6, 14). A mutation of human Grhl2 results in progressive gulated by extracellular signal-regulated kinase (ERK) phosphory- hereditary deafness, which is presumably due to defective epi- lation, which is required for wound-dependent expression of GRH thelia in the cochlea, where Grhl2 is abundantly expressed (15). target genes in epidermal cells. Serine 91 is the principal residue in Drosophila GRH is a transcription factor that can bind to DNA GRH that is phosphorylated by ERK. Although mutations of the regulatory elements of Ddc and Ultrabithorax (Ubx) (16) and is ERK phosphorylation sites in GRH do not impair its DNA binding encoded by the grainy head (grh) gene, which was originally de- function, the ERK sites in GRH are required to activate Dopa decar- fined by mutations that result in weak larval cuticle (6). The GRH boxylase (Ddc) and misshapen (msn) epidermal wound enhancers protein has a transactivation domain in the N terminus and DNA as well as functional regeneration of an epidermal barrier upon binding and dimerization domains in the C terminus of the pro- wounding. This result indicates that the phosphorylation sites are fi tein (17, 18) (Fig. 1A). High-af nity DNA binding of GRH BIOLOGY essential for damaged epidermal barrier repair. However, GRH requires homodimerization and induces activation or repression with mutant ERK phosphorylation sites can still promote barrier of GRH-dependent target gene transcription (17, 19, 20). How- DEVELOPMENTAL formation during embryonic epidermal development, suggesting ever, we still have very limited information on how the tran- that ERK sites are dispensable for the GRH function in establishing scriptional activity of GRH is regulated in cells and organisms. epidermal barrier integrity. These results provide mechanistic in- During Drosophila development, alternative splicing of grh sight into how tissue repair can be initiated by posttranslational transcripts generates two major protein isoforms, GRH-O and modification of a key transcription factor that normally mediates GRH-N (21). GRH-O is expressed in regions of the central the developmental generation of that tissue. nervous system (CNS), whereas GRH-N is expressed in barrier epithelia of the epidermis, foregut, hindgut, and tracheal system. embryo | Drosophila | cuticle Zygotic grh mutants die at the embryonic/larval transition with weak epidermal cuticle and discontinuous grainy sclerites in the nimals produce a protective epidermal barrier against head skeleton due to, in part, a lack of Ddc gene activation, a key Aphysical, chemical, and thermal damage, as well as dehy- enzyme involved in cuticular sclerotization (6). Clones of grh dration and pathogen infection. When the barrier is damaged, it mutant cells in the adult epidermis have defects in pigmentation is essential for animals to repair and regenerate the wounded and planar polarity (22). In addition, the mutation of grh leads to barrier structure to survive in a hostile environment. Biological a tortuous morphology of tracheal tubes (23). These abnormal- surface barrier function is conferred by epidermal cells, which in ities suggest that GRH plays diverse roles in establishing and mammals and arthropods produce the stratum corneum and maintaining epithelial barriers in normal development. cuticle, respectively. The stratum corneum consists of layers of We have shown that grh mutant embryos have abnormal repair a highly cross-linked matrix of dead keratinocytes, proteins, and of the cuticle barrier after epidermal wounding. The barrier re- lipids (1). In contrast, the cuticle barrier of Drosophila and other pair defects presumably results from a reduction of transcription insects consists of cross-linked lipids, proteins, and chitin (2). from a variety of GRH-target genes, one of which is Ddc (4). The In both Drosophila and mouse, transcription factors of the MAP kinase ERK is strongly activated in epidermal cells sur- Grainy head (GRH) family have been shown to be essential for rounding wounded sites, and ERK inhibition silences GRH- the development of epithelial barriers as well as the repair of mediated Ddc expression around epidermal wounds (4). From barriers after wounding (3–8). There is also evidence from this result, we inferred that ERK is in the signaling pathway from studies in Caenorhabditis elegans, Xenopus laevis, and Danio rerio injury to activation of GRH-dependent wound enhancers. It was indicating that GRH proteins have an evolutionarily conserved shown that GRH activity is controlled by the Breathless Re- role in the development and maintenance of epidermal barrier ceptor Tyrosine Kinase in the embryonic trachea (23) and that structure (9–11). All three murine homologs of GRH, Grainy GRH can be phosphorylated by ERK in vitro (20). However, head-like (Grhl) 1–3, are highly expressed in developing and there has been a lack of evidence supporting a direct relationship differentiated mouse epidermis (12). Although the mutation of murine Grhl1 results in mild defects in epidermal development and differentiation (13), mutations in Grhl3 gene results in se- Author contributions: M.K. and W.M. designed research; M.K. performed research; M.K. vere epidermal defects including inadequate skin barrier and contributed new reagents/analytic tools; M.K. and W.M. analyzed data; and M.K. and deficient wound repair, ultimately causing lethality at birth. This W.M. wrote the paper. phenotype is due in part to reduced expression of epidermal The authors declare no conflict of interest. protein cross-linking enzymes and cell adhesion proteins (3, 7). *This Direct Submission article had a prearranged editor. Analogous to the role of Grhl1 and Grhl3 proteins in mouse, 1To whom correspondence should be addressed. E-mail: [email protected]. GRH protein in Drosophila activates genes such as Dopa This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. decarboxylase (Ddc). Ddc is required to produce the quinones 1073/pnas.1016386108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1016386108 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 A S88PDS91P TAD DBD DD SST WT 1063 AA 2A 1063 AAA AAA AAAAAAAAAAA Fig. 1. ERK phosphorylation sites in GRH protein. (A) Dia- PanA 1063 grams of wild-type and deletion mutant GRH proteins: N1 603 TAD, transcriptional activation domain; DBD, DNA binding N2 136 domain; DD, dimerization domain. Numbers indicate the N3 74 C-terminal residue of each protein. (B) Purified ERK was incubated with no substrates (lane C), fusion proteins con- CMN1WTN2 N3 BPCMN1WTN2 N3 BP B (kDa) C 10 taining wild-type (WT) GRH, deletion mutants GRH-N1, GRH-N2, and GRH-N3, or a positive control substrate (My- 190 8 γ 32 120 elin Basic Protein, MBP) in the presence of [ - P] ATP. Molecular size markers (kilodaltons) are indicated. Left 85 6 60 shows Coomassie blue-stained gel, and red dotted circles 50 4 indicate individual recombinant GRH proteins or the posi- tive control substrate. Right shows 32P-autoradiography, 40 2 and blue dotted circles indicate the positions of the GST- GRH and MBP proteins. (C) Relative phosphorylation of GRH Relative Phosphorylation Relative 0 25 by ERK quantified by densitometry. The bar graph indicates WT N1 N2 N3 MBP 20 Protein 32P the mean of three independent experiments, and error bars indicate SDs. (D) GRH S91A mutant displays a strong re- A A A duction in the in vitro phosphorylation by ERK compared D 8 1 E n T 8 9 T A a with GRH WT. (E) GRH Pan Ala (PanA) mutation abolishes C W S S C W 2 P ERK-dependent phosphorylation compared with GRH WT 32P-GRH 32P-GRH and GRH S88A/S91A (2A) mutant. The data presented in this figure are representative of three independent experi- 32 GRH GRH ments. In D and E, Upper ( P-GRH) show autoradiographs and Lower (GRH) show Coomassie blue-stained gels. between ERK and GRH in late embryonic epidermal tissue and lation of the GRH 2A mutant suggested the existence of addi- the physiological importance of GRH phosphorylation in epi- tional minor phosphorylation sites. Therefore, we replaced all S dermal barrier generation and regeneration. To explore these or T residues in putative ERK phosphorylation sites (S-P or T- relationships, we first mapped ERK phosphorylation sites in the P), with A (GRH PanA mutant; Fig. 1A). The GRH PanA GRH-N protein. The identified sites were mutated to generate mutant protein is not detectably phosphorylated by ERK in vitro versions of GRH-N that had little or no phosphorylation by (Fig. 1E and Fig. S1B). Taken together, these data indicate that ERK. These wild-type and mutant forms of GRH-N were ex- ERK efficiently phosphorylates GRH on S-91, and phosphor- pressed in the epidermis of transgenic embryos that had no en- ylates other S-P and T-P sites such as S-88 with lower efficiency. dogenous GRH. To our surprise, we found that ERK phos- phorylation sites in GRH-N are dispensable for developmental Mutations of ERK Phosphorylation Sites in GRH Do Not Impair Its DNA establishment of epidermally derived cuticular barriers and ex- Binding Function in Vitro.