The cDNA of a novel, ubiquitously expressed protein kinase (Dyrk) was cloned from a rat brain cDNA library. The deduced amino acid sequence (763 amino acids) contains a catalytic domain that is only distantly related to that of other mammalian protein kinases. Its closest relative is the protein kinase Mnb of Drosophila, which is presumably involved in postembryonic neurogenesis (85% identical amino acids within the catalytic domain). Outside the catalytic domain, the sequence comprises several striking structural features: a bipartite nuclear translocation signal, a tyrosine-rich hydrophilic motif flanking the nuclear localization signal, a PEST region, a repeat of 13 histidines, a repeat of 17 serine/threonine residues, and an alternatively spliced insertion of nine codons. A recombinant glutathione S-transferase-Dyrk fusion protein catalyzed autophosphorylation and histone phosphorylation on tyrosine and serine/threonine residues with an apparent Km of approximately 3.4 microM. Exchange of two tyrosine residues in the "activation loop" between subdomains VII and VIII for phenylalanine almost completely suppressed the activity and tyrosine autophosphorylation of Dyrk. Tyrosine autophosphorylation was also reduced by exchange of the tyrosine (Tyr-219) in a tyrosine phosphorylation consensus motif. The data suggest that Dyrk is a dual specificity protein kinase that is regulated by tyrosine phosphorylation in the activation loop and might be a component of a signaling pathway regulating nuclear functions.

The cDNA of a novel protein kinase (referred to as SNRK) was isolated from a rat fat cell cDNA library with a probe generated by a cloning approach based on the polymerase chain reaction. The encoded polypeptide (746 amino acids, M, = 8 1627) contains all conserved subdomains characteristic of the protein serhelthreonine kinase family. A recombinant fusion protein with glutathionc S-transferase catalysed autophosphorylation as well as phosphorylation of histone, confirming that SNRK has indeed protein kinase activity. By Northern blot hybridization, a 5-kb mRNA was detected in brain, heart, fat cells, intestine, testis, ovary, adrenal gland and thymus. In 3T3-LI cells, SNRK was specifically expressed in the differentiated, adipocyte-like phenotype, whercas its mRNA was not detected in fibroblasts. Sequence comparisons of its catalytic domain relate SNRK to the SNFl family of protein kinases. The noncatalytic domain comprises several intriguing structural features, including a glycine-rich region, two PEST sequences, and a bipartite nuclear localization signal which is preceded by a stretch of ten consecutive acidic residues. This part of the sequence exhibits no extended similarity with other protein addition, we detected ii high degree of sequence similarity with other SNFl-related protein kinases in a small region (30-35 amino acids) flanking the C-terminus of the catalytic domain. This domain (designated the SNH domain) appears to define the subfamily of SNFI-related protein kinases and might represent a new type of regulatory domain of protein kinases.

A cDNA clone of a protein kinase with high similarity to the Clk (Cdc2-like kinases) subfamily was isolated from a rat brain library and characterized. Its deduced amino acid sequence exhibited a 99% identity with human Clk3 and was therefore designated rat Clk3. In addition to the protein kinase domain, the sequence (490 amino acids) comprises an N-terminal domain with a strikingly high portion of basic amino acids. A glutathione S-transferase fusion protein of Clk3 catalyzed autophosphorylation of the kinase but not phosphorylation of the exogenous substrates histone or casein. By Northern blot analysis of different rat tissues, mRNA of Clk3 was detected predominately in testis, suggesting that this kinase regulates a predominately testicular function.