Detection of an Appropriate Kinase Activity in Branchial Arches I and II That Coincides with Peak Expression of the Treacher Collins Syndrome Gene Product, Treacle

Natalie C. Jones 0 Peter G. Farlie 0 Joe Minichiello 0 Don F. Newgreen 0 0 Murdoch Institute, Royal Children's Hospital , Flemington Road, Parkville, Victoria 3052, Australia Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder involving the mid and lower face and, in particular, the tissues affected arise solely from embryonic branchial arches I and II. TCOF1, the gene involved in TCS, has been cloned and although the function of the encoded protein, treacle, has not yet been established, it exhibits peak expression in the branchial arches. Treacle contains a series of repeating units of acidic and basic residues, which are predicted to contain putative casein kinase II (CKII) and protein kinase C (PKC) phosphorylation site motifs. In addition, treacle has weak homology to two phosphorylation-dependent nucleolar proteins, which shuttle between the cytoplasm and nucleolus. Based on these observations, phosphorylation of treacle may be important for its function. In this study, GST-treacle fusion peptides were constructed using particular TCOF1 exons that contained potential CKII and PKC phosphorylation sites. These were used as substrates in in vitro kinase assays and showed that treacle fusion peptides can be phosphorylated by the appropriate kinases. Furthermore, using tissue extracts we have demonstrated that in avian embryonic branchial arches I and II there is a kinase activity that can phosphorylate treacle peptides that is consistent with CKII site recognition. This activity coincides with the reported high expression of treacle in these tissues at early developmental stages and declines later in development. - Treacher Collins syndrome (TCS), an autosomal dominant disorder of craniofacial development, occurs with an incidence of 1/50 000 live births (1,2). While 40% of TCS cases have a previous family history, 60% of cases possibly arise as a result of a de novo mutation (3). The clinical features of TCS are generally symmetrical (4) and include: (i) abnormalities of the external ears, narrowing of the external ear canals and malformation of the middle ear ossicles, which may lead to conductive hearing loss (5); (ii) lateral downward sloping of the palpebral fissures with colobomas of the lower eyelids and lack of lower eyelashes; (iii) hypoplasia of the mandible and zygomatic complex and cleft palate (1,6). TCS displays high penetrance, with expression of the clinical phenotype and severity of the disorder showing inter- and intrafamilial variability (7). The TCS gene, TCOF1, was identified via positional cloning (811) and found to encode a low complexity, serine/alaninerich protein which was named treacle (12,13). Analysis of this gene in TCS affected individuals identified over 50 diseasecausing mutations that were spread throughout TCOF1, with the majority of the mutations introducing a premature termination codon (1215). Although the TCOF1 gene has been cloned, the precise function and biochemical nature of the encoded protein has not been determined. There are 10 repeating units containing clusters of acidic amino acids that are separated by basic amino acid residues within treacle (7). Each repeating unit is confined to a single exon and comparison against pattern databases predicts that these repeat units contain putative casein kinase II (CKII) and protein kinase C (PKC) phosphorylation site motifs (7). An additional feature of treacle is the identification of potential nuclear localization signals (NLSs) near the C-terminus (7). Recent studies have demonstrated that these NLSs enable treacle to be targeted to the nucleolus (16,17). Database comparisons have revealed weak homology between treacle and two phosphorylation-dependent nucleolar phosphoproteins, Xenopus laevis nucleolar phosphoprotein and rat nucleolar phosphoprotein 140 (Nopp140) (7,18,19). These nucleolar phosphoproteins shuttle between the nucleolus and cytoplasm, suggesting a role as chaperones in protein transport (7,19). All three proteins appear to be low complexity proteins, with a majority of the same five amino acids (i.e. serine, alanine, lysine, proline and glutamic acid). Furthermore, the sequence similarity between treacle and the nucleolar phosphoproteins is greatest at the repeating motifs, where the potential sites for CKII phosphorylation are predicted. The similarity of treacle to nucleolar phosphoproteins such as Nopp140 suggests that treacle may function in protein import or export by shuttling between the nucleolus and cytoplasm. This hypothesis is supported by recent work which showed that mutations in TCOF1 cause the resulting truncated protein to become mislocalized within the cell (16). +These authors contributed equally to this work To whom correspondence should be addressed. Tel: +61 3 8341 6243; Fax: +61 3 9348 1391; Email: On the basis of the TCS phenotype, treacle must play a fundamental role in embryonic development. The craniofacial defects observed in TCS affect tissues derived from branchial arches I and II, which form during the first 4 weeks of human development (20). Since neural crest cells contribute significantly to formation of the branchial arches, dysmorphogenesis of the lower face may therefore be a consequence of defective neural crest cell migration from the hindbrain to the branchial arches during early embryogenesis (21,22) and/or neural crest cell survival, proliferation, differentiation or metabolism. Hence, knowledge of the processes involved in development of the branchial arches or their derivatives may lead to identification of a function for the treacle protein. Further analysis was achieved through isolation of the murine homologue of the gene involved in TCS, tcof1. (15). tcof1 has a slightly smaller open reading frame than human TCOF1, but it also encodes a low complexity, serine/alanine-rich protein (15). Observations from expression analysis in the mouse support the suggestion that the TCS gene is involved in development of the craniofacial complex during embryogenesis (15), since it is highly expressed in the early branchial arches, while also being expressed at lower concentrations at other sites and stages. In our current investigation, in vitro kinase assays were used to demonstrate whether glutathione S-transferase (GST) treacle fusion peptides containing potential CKII and PKC phosphorylation motifs could be phosphorylated by the appropriate kinases. Subsequent experiments using crude tissue extracts derived from various avian tissues, including branchial arches, showed that a kinase activity that could use a specific treacle peptide as a substrate was expressed in the appropriate temporal pattern (i.e. during early embryonic development). Thus, this kinase activity broadly coincides with peak expression of treacle (i.e. at early stages in branchial arches I and II), which is consistent with phosphorylation playing a role in the biology of treacle. Construction of GSTtreacle fusion pepti (...truncated)