We have developed a method to study the primary sequence specificities of protein kinases by using an oriented degenerate peptide library. We report here the substrate specificities of eight protein Ser/Thr kinases. All of the kinases studied selected distinct optimal substrates. The identified substrate specificities of these kinases, together with known crystal structures of protein kinase A, CDK2, Erk2, twitchin, and casein kinase I, provide a structural basis for the substrate recognition of protein Ser/Thr kinases. In particular, the specific selection of amino acids at the ؉1 and ؊3 positions to the substrate serine/threonine can be rationalized on the basis of sequences of protein kinases. The identification of optimal peptide substrates of CDK5, casein kinases I and II, NIMA, calmodulin-dependent kinases, Erk1, and phosphorylase kinase makes it possible to predict the potential in vivo targets of these kinases.The essential role of protein kinases in regulating signal transduction was established with the discovery of cyclic AMPdependent protein kinase (PKA) (12). To respond to different extracellular stimuli, distinct groups of protein kinases have evolved. Each protein kinase is thought to phosphorylate a unique set of targets in the cell. The substrate specificities of protein kinases are therefore crucial for the fidelity of signaling events.The classical approach for studying the specificity of a protein kinase is to compare the phosphorylation kinetics of synthetic peptides on the basis of known sequences phosphorylated by the kinase. This procedure is helpful in identifying the amino acids critical for efficient phosphorylation. However, it is not practical to synthesize and study each of the billions of possible variations of sequences that must be considered. Moreover, it is extremely difficult to apply this approach to study the specificity of a protein kinase with no known substrates. To overcome these problems, we developed a method for determining the primary sequence specificities of protein kinases by using an oriented degenerate peptide library (21). Optimal peptide substrates of a given protein kinase are identified by phosphorylation of a pool of degenerate peptides containing billions of different species. The specificities determined for PKA, CDC2, and CDK2 by using this technique were consistent with known substrates of these kinases. The results also allowed the prediction of in vivo kinase substrates. Synthetic peptides based on predicted optimal motifs were shown to act as low-K m substrates for the kinases studied. Therefore, this method is a useful tool for studying substrate specificities of protein kinases.We present here the specificities of eight additional protein Ser/Thr kinases: CDK5, casein kinase I (CKI) ␦ and ␥, casein kinase II (CKII), NIMA, calmodulin-dependent (Cam) kinase II, Erk1, and phosphorylase kinase. Our findings demonstrate that each of these protein kinases has a distinct optimal peptide substrate. Critical determinants for recognition by the protein kinas...

Presynaptic inhibition mediated by G protein-coupled receptors may involve a direct interaction between G proteins and the vesicle fusion machinery. The molecular target of this pathway is unknown. We demonstrate that Gbetagamma-mediated presynaptic inhibition in lamprey central synapses occurs downstream from voltage-gated Ca(2+) channels. Using presynaptic microinjections of botulinum toxins (BoNTs) during paired recordings, we find that cleavage of synaptobrevin in unprimed vesicles leads to an eventual exhaustion of synaptic transmission but does not prevent Gbetagamma-mediated inhibition. In contrast, cleavage of the C-terminal nine amino acids of the 25 kDa synaptosome-associated protein (SNAP-25) by BoNT A prevents Gbetagamma-mediated inhibition. Moreover, a peptide containing the region of SNAP-25 cleaved by BoNT A blocks the Gbetagamma inhibitory effect. Finally, removal of the last nine amino acids of the C-terminus of SNAP-25 weakens Gbetagamma interactions with soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Thus, the C terminus of SNAP-25, which links synaptotagmin I to the SNARE complex, may represent a target of Gbetagamma for presynaptic inhibition.

The activation of G protein-coupled receptors (GPCRs) can result in an inhibition of Ca(2+)-dependent hormone and neurotransmitter secretion. This has been attributed in part to G protein inhibition of Ca(2+) influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca(2+) entry. Here we characterize direct inhibitory actions of G protein betagamma (Gbetagamma) on Ca(2+)-triggered vesicle exocytosis in permeable PC12 cells. Gbetagamma inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated protein of 25 kD (SNAP25). Gbetagamma bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca(2+)-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gbetagamma interactions with the Ca(2+)-dependent fusion machinery.

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ABSTRACT:Cytochrome P450 (P450) 20A1 is one of the so-called "orphan" P450s without assigned biological function. mRNA expression was detected in human liver, and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3-to 4-fold higher in brain and heart than in liver. In situ hybridization of rat whole brain sections showed an mRNA expression pattern similar to that observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200-280 nmol of P450/ l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function.P450 monooxygenases catalyze the introduction of oxygen into a vast range of molecules and are known to have diverse functions in endogenous and exogenous metabolism (Ortiz de Montellano, 2005). Cytochromes P450 (P450s) in families 1 to 3 are involved primarily in the metabolism of exogenous compounds, i.e., drugs and environmental pollutants, whereas families 4 to 51 consist of enzymes involved primarily in the bioconversion of endogenous compounds, i.e., steroids, fatty acids, vitamins, and eicosanoids (Guengerich, 2005). To date 57 human P450 (CYP or P450, P450 indicating the nucleotide or protein and CYP indicating the gene in question) genes are known (http://drnelson.utmem.edu/CytochromeP450.html). The P450s can be divided into six major groups based on their main substrates: steroids, vitamins, fatty acids, eicosanoids, xenobiotics, and unknown (Nelson et al., 1996;Guengerich et al., 2005). It is noteworthy, however, that a number of known P450s have assigned substrates in more than one of these groups, and some substrates cannot be assumed to be true physiological substrates based only on their conversion rates (e.g., testosterone 6␤-hydroxylation for hepatic P450 3A4), in the absence of other evidence of function. Although extensive research efforts have been directed to elucidating the endogenous and exogenous functions of individual P450s, one-fourth of the human P450s still have not been assigned ...

Glycogen phosphorylase is found in resting muscle as phosphorylase b, which is inactive without AMP. Phosphorylation by phosphorylase kinase (PhK) produces phosphorylase a, which is active in the absence of AMP. PhK is the only kinase that can phosphorylate phosphorylase b, which in turn is the only physiological substrate for PhK. We have explored the reasons for this specificity and how these two enzymes recognize each other by studying site-directed mutants of glycogen phosphorylase. All mutants were assayed for changes in their interaction with a truncated form of the catalytic subunit of phosphorylase kinase, gamma(1-300). Five mutations (R69K, R69E, R43E, R43E/R69E, and E501A), made at sites that interact with the amino terminus in either phosphorylase b or a, showed little difference in phosphorylation by gamma(1-300) compared to wild-type phosphorylase b. Five mutations, made at three sites in the amino-terminal tail of phosphorylase (K11A, K11E, I13G, R16A, and R16E), however, produced decreases in catalytic efficiency for gamma(1-300), compared to that for phosphorylase b. R16E was the poorest substrate for gamma(1-300), giving a 47-fold decrease in catalytic efficiency. The amino terminus, and especially Arg 16, are very important factors for recognition of phosphorylase by gamma(1-300). A specific interaction between Lys 11 of phosphorylase and Glu 110 of gamma(1-300) was also confirmed. In addition, I13G and R16A were able to be phosphorylated by protein kinase A, which does not recognize native phosphorylase.

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