title: Phosphorylation by LAMMER protein kinases: determination of a consensus site, identification of in vitro substrates, and implications for substrate preferences.
abstract: LAMMER protein kinases are ubiquitous throughout eukaryotes, including multiple paralogues in mammals. Members are characterized by similar overall structure and highly identical amino acid sequence motifs in catalytic subdomains essential for phosphotransfer and interaction with substrates. LAMMER kinases phosphorylate and regulate the activity of the SR protein class of pre-mRNA splicing components, both in vitro and in vivo. In this study, we define an optimum in vitro consensus phosphorylation site for three family members using an oriented degenerate peptide library approach. We also examine the substrate specificity and interactions of several LAMMER protein kinases from widely diverged species with potential substrates, including their own N-termini, predicted to be substrates by the peptide-based approach. Although the optimal in vitro consensus phosphorylation site for these kinases is remarkably similar for short peptides, distinct substrate preferences are revealed by in vitro phosphorylation of intact proteins. This finding suggests that these kinases may possess varied substrates in vivo, and thus the multiple LAMMER kinases present in higher eukaryotes may perform differentiable functions. These results further demonstrate that these kinases can phosphorylate a number of substrates in addition to SR proteins, suggesting that they may regulate multiple cellular processes, in addition to the alternative splicing of pre-mRNAs.
authors.affiliation: Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, 54 006 Thessaloniki, Greece.
authors.forename: Eleni
authors.initials: E
authors.lastname: Nikolakaki
authors.forename: Cheng
authors.initials: C
authors.lastname: Du
authors.forename: Jack
authors.initials: J
authors.lastname: Lai
authors.forename: Thomas
authors.initials: T
authors.lastname: Giannakouros
authors.forename: Lew
authors.initials: L
authors.lastname: Cantley
authors.forename: Leonard
authors.initials: L
authors.lastname: Rabinow
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D016384:Consensus Sequence; D018076:DNA, Complementary / Q000235:genetics; D029721:Drosophila Proteins; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010940:Plant Proteins; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
chemical_list: D018076:DNA, Complementary; D029721:Drosophila Proteins; D010455:Peptides; D010940:Plant Proteins; C103792:PK12 protein, Nicotiana tabacum; D011494:Protein Kinases; C066780:Clk dual-specificity kinases; C089977:Doa protein, Drosophila; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases