Mutations at the Darkener of apricot (Doa) locus of Drosophila cause roughened eyes and increase transcript accumulation from the retrotransposon copia up to fourfold. Cloning of the gene and sequencing of cDNAs reveals that it encodes a putative serine/threonine protein kinase. Sequence data base searches identify it is a member of a novel highly conserved protein kinase family, with homologs in humans, mice, and Saccharomyces cerevisiae, not related to each other previously. Family members are characterized by a peptide motif reading EHLAMMERILG at kinase subdomain X, which is virtually 100% identical in all homologs. We therefore refer to this new family as the LAMMER protein kinases. As predicted from its primary sequence, Doa protein possesses intrinsic protein kinase activity when expressed in bacteria, as assayed via autophosphorylation. The gene is expressed throughout development, and both stage and tissue-specific RNAs are found. Its function is essential, because maternally deposited or zygotically transcribed mRNA is required for development to larval stages, and defects in segmentation and development of the nervous system are observed in embryos derived from heteroaUelic mothers. Doa function is also critical to Drosophila eye development, because the organization and development of pigment cells, bristles, and photoreceptors are affected in various mutant classes. In the most extreme cases that survive to adulthood, retinal photoreceptors degenerate prior to eclosion. These results demonstrate that the kinase encoded by Doa is required at multiple stages of development, for both differentiation and maintenance of specific cell types.

Nutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism.

Alternative mRNA splicing directed by SR proteins and the splicing regulators TRA and TRA2 is an essential feature of Drosophila sex determination. These factors are highly phosphorylated, but the role of their phosphorylation in vivo is unclear. We show that mutations in the Drosophila LAMMER kinase, Doa, alter sexual differentiation and interact synergistically with tra and tra2 mutations. Doa mutations disrupt sex-specific splicing of doublesex pre-mRNA, a key regulator of sex determination, by affecting the phosphorylation of one or more proteins in the female-specific splicing enhancer complex. Examination of pre-mRNAs regulated similarly to dsx shows that the requirement for Doa is substrate specific. These results demonstrate that a SR protein kinase plays a specific role in developmentally regulated alternative splicing.

Clk/STY, the murine homologue of the recently described LAMMER family of protein kinases, autophosphorylates on serine/threonine and tyrosine residues in vitro and in vivo. LAMMER kinases are found throughout eukaryotes and possess virtually complete amino acid identity in many domains critical for kinase function, leading to the question of whether other family members also possess dual specificity. We report here that the Drosophila family member DOA, human SK-G1, and the Saccharomyces cerevisiae KNS1, all possess protein kinase activity and autophosphorylate with dual specificity in vitro, suggesting that the entire family possesses this activity. Although the LAMMER kinases are closely related to the mitogen-activated protein kinase family, they possess different substrate specificity in vitro, based on phosphorylation of peptide and protein substrates and sequencing of a phosphorylation site in a common substrate.Protein kinases are generally differentiated according to the specificity with which they phosphorylate substrates, with activity toward serine/threonine or tyrosine residues (1, 2). Recently, some kinases capable of phosphorylating all three amino acid residues have been identified (reviewed in Refs. 3 and 4). Such "dual specificity" kinases would be classified as serine/threonine-specific based on their amino acid sequences. Among these is murine STY or Clk (5, 6), whose cDNAs were isolated because their products autophosphorylated on tyrosine residues. Data base searches with the sequence of cDNAs from the Doa locus of Drosophila revealed murine Clk and other closely related homologues in eukaryotes ranging from yeast to humans (7). Additional homologues have subsequently been described in green plants (8) 1 (also accession number D49304 from the rice genome sequencing project), from rat (accession number X94351), and from Schizosaccharomyces pombe (accession number Z69239), a third family member in humans (9), a mink family member whose message is induced by cycloheximide in cultured cells (10), and a member from S. pombe, found in the genome sequencing project of this organism (accession number Z69239). We have dubbed these the LAMMER protein kinases, based upon the existence of this motif, or conserved variations upon it, in all members (7).The LAMMER kinases are nearly identical in size, spacing, and placement of their kinase catalytic domains and also show extremely high amino acid identity in domains essential for phosphotransfer to the peptide substrate and in substrate recognition (11). By analogy with the structure of two crystallized protein kinases (12, 13), the LAMMER motif lies in an ␣-helix below the substrate-binding cleft, potentially allowing it to make contact with substrates, and suggesting that these molecules are also highly conserved. Much of what is known of LAMMER kinase function derives from analysis of the Drosophila locus Doa, whose mutants were isolated during screens for loci with transcriptional regulatory effects (14,15). Doa is an essential gene; its mutations alt...