Biomedical subjects

S S Taylor

Publications and source records attributed to S S Taylor.

At least 19 recordsLinked to original sources

PubMed1995

Tyrosines outside the kinase core and dimerization are required for the mitogenic activity of RET/ptc2.

Defects in the c-ret proto-oncogene, a member of the protein tyrosine kinase receptor family, have recently been linked to two types of genetic syndromes, Hirschsprung's disease and the multiple endocrine neoplasia family of inherited cancers. RET/ptc2 is the product of a papillary thyroid carcinoma translocation event between the genes coding for c-ret and the type I alpha regulatory subunit of protein kinase A (RI alpha) (Lanzi, C., Borrello, M., Bongarzone, I., Migliazza, A., Fusco, A., Grieco, M., Santoro, M., Gambetta, R., Zunino, F., Della Porta, G., and Pierotti, M. (1992) Oncogene 7, 2189-2194). The resulting 596-residue protein contains the first two-thirds of RI alpha and the entire tyrosine kinase domain of c-ret (RETtk). An in vivo assay of growth stimulatory effects was developed, which consisted of microinjecting a RET/ptc2 expression plasmid into the nuclei of 10T1/2 mouse fibroblasts and observing the incorporation of 5-bromodeoxyuridine. This assay was used to determine that only the dimerization domain of RI alpha fused to RETtk is required for RET/ptc2's mitogenic activity. In addition, all of the reported Hirschsprung's disease point mutations in the RETtk (S289P, R421Q, and R496G) inactivate RET/ptc2 in our assay, confirming that these are loss of function mutations. Two tyrosines outside the conserved kinase core were also identified that are essential for full mitogenic activity of RET/ptc2. These two tyrosines, Tyr-350 and Tyr-586, are potential sites for Src homology 2 and phosphotyrosine binding domain interactions.

Animals↗

PubMed1995

Identification of a signal for rapid export of proteins from the nucleus.

Active nuclear import of protein is controlled by nuclear localization signals (NLSs), but nuclear export is not understood well. Nuclear trafficking of the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) is critical for regulation of gene expression. The heat-stable inhibitor (PKl) of cAPK contains a nuclear export signal (NES) that triggers rapid, active net extrusion of the C-PKl complex from the nucleus. This NES (residues 35-49), fused or conjugated to heterologous proteins, was sufficient for rapid nuclear export. Hydrophobic residues were critical. The NES is a slightly weaker signal than the SV40 NLS. A sequence containing only residues 37-46, LALKLAGLDI, is also sufficient for nuclear export. This is an example of a protein-based NES having no obvious association with RNA. A similar sequence, LQLPPLERLTL, from Rev, an RNA-binding protein of HIV-1, also is an NES.

Amino Acid Sequence↗

PubMed1995

Regulatory subunit of protein kinase A: structure of deletion mutant with cAMP binding domains.

In the molecular scheme of living organisms, adenosine 3',5'-monophosphate (cyclic AMP or cAMP) has been a universal second messenger. In eukaryotic cells, the primary receptors for cAMP are the regulatory subunits of cAMP-dependent protein kinase. The crystal structure of a 1-91 deletion mutant of the type I alpha regulatory subunit was refined to 2.8 A resolution. Each of the two tandem cAMP binding domains provides an extensive network of hydrogen bonds that buries the cyclic phosphate and the ribose between two beta strands that are linked by a short alpha helix. Each adenine base stacks against an aromatic ring that lies outside the beta barrel. This structure provides a molecular basis for understanding how cAMP binds cooperatively to its receptor protein, thus mediating activation of the kinase.

Affinity Labels↗

PubMed1995

The expression and intracellular distribution of the heat-stable protein kinase inhibitor is cell cycle regulated.

The heat-stable protein kinase inhibitor (PKI) is a potent and specific inhibitor of the catalytic (C) subunit of the cAMP-dependent protein kinase. We report the isolation of a polyclonal antibody raised to purified recombinant PKI alpha. Using this antibody, the intracellular distribution of endogenous PKI alpha was assessed by immunostaining. The PKI alpha expression and intracellular distribution varied as a function of cell cycle progression. PKI alpha expression appeared low in serum-starved cells and in cells in G1 and increased as cells progressed through S phase. Its distribution became increasingly nuclear as cells entered G2/M. Nuclear levels of PKI alpha remained high through cell division and decreased again as cells reentered G1. The cell cycle regulated expression and nuclear distribution suggests a specific role for PKI alpha in the nucleus during the G2/M phases of the cell cycle. Consistent with this, microinjection of PKI alpha antibody into serum-starved cells prevented their subsequent cell cycle progression. Similarly, overexpression of C subunit in cells arrested at the G1/S boundary prevented their subsequent division. Together these results support the idea that PKI alpha plays an important role in the inhibition of nuclear C subunit activity required for cell cycle progression, although a determination of the relative amounts of endogenous nuclear PKI and C-subunit will be required to substantiate this hypothesis.

Animals↗

PubMed1995

How do protein kinases discriminate between serine/threonine and tyrosine? Structural insights from the insulin receptor protein-tyrosine kinase.

The eukaryotic protein kinases that directly phosphorylate proteins are divided into two major classes: those that phosphorylate tyrosine and those that phosphorylate serine and threonine. Until recently, the similarities between these two classes of enzymes, which now total more than 400, were based primarily on sequence alignments. A recent report of the structure of the kinase domain (IRK) of the insulin receptor protein-tyrosine kinase now allows the features of these two families to be compared at the structural level. We review here this first tyrosine-specific protein kinase structure, and compare and contrast it to the structure of the serine/threonine-specific cAMP-dependent protein kinase. Although the general fold of the polypeptide backbone is conserved as predicted, unique features at the IRK active site provide a basis for understanding the differences in specificity for the phosphate acceptor amino acid. The structure of this inactive, dephosphorylated protein-tyrosine kinase also defines for the first time how activation might be achieved.

Adenosine Triphosphate↗

PubMed1995

Two highly similar multidrug transporters of Bacillus subtilis whose expression is differentially regulated.

The Bacillus subtilis genome encodes two multidrug efflux transporters sharing 51% sequence identity: Bmr, described previously, and Blt, described here. Overexpression of either transporter in B. subtilis leads to a similar increase in resistance to ethidium bromide, rhodamine and acridine dyes, tetraphenylphosphonium, doxorubicin, and fluoroquinolone antibiotics. However, Blt differs widely from Bmr in its expression pattern. Under standard cultivation conditions, B. subtilis expresses Bmr but Blt expression is undetectable. We have previously shown that Bmr expression is regulated by BmrR, a member of the family of MerR-like transcriptional activators. Here we show that blt transcription is regulated by another member of the same family, BltR. The DNA-binding domains of BmrR and BltR are related, but their putative inducer-binding domains are dissimilar, suggesting that Bmr and Blt are expressed in response to different inducers. Indeed, rhodamine, a substrate of Bmr and Blt and a known inducer of Bmr expression, does not induce Blt expression. Blt expression has been observed only in B. subtilis, carrying mutation acfA, which, as we show here, alters the sequence of the blt gene promoter. Unlike bmr, which is transcribed as a monocistronic mRNA, blt is cotranscribed with a downstream gene encoding a putative acetyltransferase. Overall, the differences in transcriptional control and operon organization between bmr and blt suggest that the transporters encoded by these genes have independent functions involving the transport of distinct physiological compounds.

Acetyltransferases↗

PubMed1994

Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal.

The heat-stable inhibitor of cAMP-dependent protein kinase (PKI) was shown previously to export the kinase catalytic subunit (C) from the nucleus (Fantozzi, D. A., Harootunian, A. T., Wen, W., Taylor, S. S., Feramisco, J.R., Tsien, R. Y., and Meinkoth, J. L. (1994) J. Biol. Chem. 269, 2676-2686), in addition to its ability to inhibit kinase activity. In this study, the mechanism of PKI export is investigated. The injection of a C-PKI complex containing both labeled PKI and C-subunit revealed that both proteins exit the nucleus in unison. A fusion protein of C-subunit with glutathione S-transferase (GST) (140 kDa) cannot transverse the nuclear membrane in either direction, but can be exported from the nucleus when complexed with PKI, supporting the presence of a nuclear export signal (NES) in the C-PKI complex. Fusions of PKI alpha with GST (70 kDa) or PKI beta 1 with maltose-binding protein (MBP) (50 kDa) remain effective at exporting complexes with C-subunit. The export of C-PKI is also sensitive to temperature and energy depletion. Taken together, these results demonstrate that export is both energy- and temperature-dependent, but size-independent, consistent with an active signal-mediated export process. GST-PKI exits from the nucleus even in the absence of C-subunit, indicating that the NES resides entirely on PKI, but suggesting that fusion of PKI to GST leads to a conformational change that mimics the exposure of the NES caused by the binding of C. Since both PKI alpha and PKI beta 1 can export C-subunit, the predicted export signal is likely to reside on the residues conserved between PKI alpha and PKI beta 1.

Adenosine Triphosphate↗

PubMed1994

Domain movements in protein kinases.

Structural studies of the catalytic subunit of the cAMP-dependent protein kinase, both by crystallographic methods and in solution, reveal two conformations. Crystal structures of several other protein kinases have also been solved in the past year. With this combined information we can begin to define mobile domains and subdomains within the conserved catalytic core.

Animals↗

PubMed1993

A conserved helix motif complements the protein kinase core.

Residues 40-300 of the mammalian catalytic (C) subunit of cAMP-dependent protein kinase define a conserved bilobal catalytic core shared by all eukaryotic protein kinases. Contiguous to the core is an extended amphipathic alpha-helix (A helix). Trp30, a prominent feature of this helix, fills a deep hydrophobic pocket between the two lobes on the surface opposite to the active site. The C subunit in Dictyostelium discoideum shows sequence conservation of residues 40-350 with the mouse enzyme but contains an N-terminal extension of 332 residues. A sequence corresponding to the A helix contiguous to the core is absent. However, we have now identified a remote A-helix motif (residues 77-98). When the core of the Dictyostelium C subunit was modeled, based on the mouse C subunit, complementarity between this putative A helix and the surface of the core was found to be conserved. Analysis of other protein kinases reveals that the A-helix motif is not restricted to cAMP-dependent protein kinase. In the Src-related family of protein kinases, for example, an A helix is very likely contiguous to the core, thus serving as a linker between the conserved catalytic core and the Src homology 2 domain. We predict that an A-helix motif complementary to the core will be a conserved feature of most eukaryotic protein kinases.

Amino Acid Sequence↗

PubMed1992

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase contains two stable phosphorylation sites, Thr-197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type II regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic pI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser-10 to phosphorylation. Either Ser-10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation remains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C alpha and C beta genes.

Adenosine Triphosphate↗

PubMed1992

DdPK3, which plays essential roles during Dictyostelium development, encodes the catalytic subunit of cAMP-dependent protein kinase.

We have previously reported the analysis of DdPK3, a developmentally regulated putative serine/threonine kinase that shares approximately 50% amino acid sequence identity with metazoan cAMP-dependent protein kinase A (PKA) and protein kinase C, within their catalytic domains. Cells in which the DdPK3 gene has been disrupted do not aggregate but they are able to induce aggregation-stage genes in response to cAMP pulses and the prestalk-specific ras gene DdrasD in response to high continuous levels of cAMP but will not induce prespore gene expression. In this report, we present conclusive evidence that DdPK3 encodes the catalytic subunit of the Dictyostelium PKA. DdPK3 null cells lack kinase activity that phosphorylates a PKA-specific substrate and is specifically inhibitable by recombinant cAMP-dependent protein kinase inhibitor. DdPK3 expressed in Escherichia coli has PKA activity that is inhibitable by protein kinase inhibitor. When Ddpk3 null cells are complemented with DdPK3 expressed from an actin promoter on an extrachromosomal vector (low copy number), PKA activity is restored and the cells proceed to the slug stage but will not culminate, suggesting that properly regulated PKA activity is essential for culmination. Moreover, overexpressing DdPK3 in wild-type cells on integrating vectors (high copy number) from either an actin or prespore-specific promoter results in accelerated development and the ability to form mature spores in monolayer culture in the presence of high cAMP, a developmental potential lacking in wild-type cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Structural basis of the intrasteric regulation of myosin light chain kinases.

The smooth muscle myosin light chain kinase (smMLCK) catalytic core was modeled by using the crystallographic coordinates of the cyclic AMP-dependent protein kinase catalytic subunit (cAPK) and a bound pseudosubstrate inhibitor peptide, PKI(5-24). Despite only 30% identity in amino acid sequence, the MLCK sequence can be readily accommodated in this structure. With the exception of the short B-helix, all major elements of secondary structure in the core are very likely conserved. The active site of the modeled MLCK complements the known requirements for peptide substrate recognition. MLCK contains a pseudosubstrate sequence that overlaps the calmodulin binding domain and has been proposed to act as an intrasteric inhibitor and occupy the substrate binding site in the absence of Ca(2+)-calmodulin. The pseudosubstrate sequence can be modeled easily into the entire backbone of PKI(5-24). The results demonstrate that the intrasteric model for regulation of MLCK by intramolecular competitive inhibition is structurally plausible.

Amino Acid Sequence↗

PubMed1992

Energetic limits of phosphotransfer in the catalytic subunit of cAMP-dependent protein kinase as measured by viscosity experiments.

Viscosogenic agents were used to test the diffusion limits of the reaction catalyzed by the catalytic subunit of the cAMP-dependent protein kinase. The effects of glycerol and sucrose on the maximum rate (kcat) and the apparent second-order rate constants (kcat/Kpeptide) for the phosphorylation of four peptidic substrates were measured at their pH optima. The agents were found to have moderate to no effect on kcat/Kpeptide for good and poor substrates, respectively. Conversely, kcat was highly sensitive to solvent viscosity for three of the four peptides at high concentrations of ATP. Taken together, these data indicate that enzymatic phosphorylation by the catalytic subunit proceeds with rapid or near rapid equilibrium binding of substrates and that all steps following the central substrate complex (i.e., chemical and conformational events) are fast relative to the rate-determining dissociation of product, ADP, when ATP levels are high. Under saturating concentrations of peptide I, LRRASLG, an unproductive form of the enzyme is populated. The observed phosphorylation rate from this complex is involved in rate limitation owing to a slow step separating unproductive and productive enzyme forms. The data are used to establish a kinetic mechanism for the catalytic subunit that predicts initial reaction velocities under varying concentrations of ATP and substrate.

Adenosine Triphosphate↗

PubMed1992

Effect of the thermostable protein kinase inhibitor on intracellular localization of the catalytic subunit of cAMP-dependent protein kinase.

cAMP-dependent protein kinase mediates a variety of cellular responses in most eukaryotic cells. Many of these responses are cytoplasmic, whereas others appear to require nuclear localization of the catalytic subunit. In order to understand further the molecular basis for subcellular localization of the catalytic subunit, the effect of the heat stable protein kinase inhibitor (PKI) was investigated. The subcellular localization of the catalytic (C) subunit was determined both in the presence and absence of PKI, by microinjecting fluorescently labeled C subunit into single living cells. When injected alone, a significant fraction of the dissociated C subunit localized to the nucleus. When coin-injected with an excess of PKI, little of the C subunit localized to the nucleus, suggesting that accumulation of catalytic subunit in the nucleus requires either enzymatic activity or a nuclear localization signal. Inactivation of the catalytic subunit in vitro by treatment with N-ethylmaleimide did not prevent localization in the nucleus, indicating that enzymatic activity was not a prerequisite for nuclear localization. In an effort to search for a specific signal that might mediate nuclear localization, a complex of the catalytic subunit with a 20-residue inhibitory peptide derived from PKI (PKI(5-24)) was microinjected. In contrast to intact PKI, the peptide was not sufficient to block nuclear accumulation. In the presence of PKI(5-24), the C subunit localized to the nucleus in a fashion analogous to that of dissociated, active C subunit despite evidence of no catalytic activity in situ. Thus, nuclear localization of the C subunit appears to be independent of enzymatic activity but most likely dependent upon a signal. The signal is apparently masked by both the regulatory subunit and PKI but not by the inhibitory peptide.

Animals↗

PubMed1992

Crystallization studies of the catalytic subunit of cAMP-dependent protein kinase: crystals of murine recombinant catalytic subunit and a mutant, Cys 343----Ser, diffract to 2.7 A resolution.

The recombinant mouse catalytic subunit of cAMP-dependent protein kinase, expressed and purified from E. coli, has been successfully cocrystallized as a binary complex with an inhibitor peptide and as a ternary complex with an inhibitor peptide and MgATP. In contrast to the catalytic subunit obtained from porcine heart, the recombinant catalytic subunit lacks a myristoyl group at the amino terminus and differs in sequence at nine positions out of 350 amino acids. The catalytic activities of the two enzymes, however, are nearly identical. Both enzymes cocrystallized with a 20-amino-acid inhibitor and MgATP; however, the porcine-heart enzyme crystallized in a hexagonal space group (P6(1)22) while the recombinant murine catalytic subunit crystallized in an orthorhombic space group (P2(1)2(1)2(1), a = 73.70, b = 76.26, c = 80.74 A). The orthorhombic crystals of the recombinant catalytic subunit exhibit the best diffraction characteristics of all catalytic subunit crystals obtained so far: 2.7 A resolution. Unlike the mammalian porcine-heart enzyme, no crystals of the recombinant apo-enzyme were obtained under the same crystallization conditions. These results are consistent with earlier conclusions that the catalytic subunit exists in at least two distinct conformational states and furthermore suggests that the inhibitor peptide alone is sufficient to induce the major conformational changes that distinguish the two states. A mutant form of the catalytic subunit where Cys343 was replaced with Ser was also cocrystallized with the 20-amino-acid peptide inhibitor and MgATP, and resulted in an orthorhombic crystal isomorphous to crystals of the unmutated enzyme with a similar diffraction of 2.7 A.

Amino Acid Sequence↗

PubMed1992

Systematic mutational analysis of cAMP-dependent protein kinase identifies unregulated catalytic subunits and defines regions important for the recognition of the regulatory subunit.

A library of mutants of the catalytic subunit of the Saccharomyces cerevisiae cAMP-dependent protein kinase was screened in vitro for mutants defective in the recognition of the regulatory subunit. The mutations identified were mapped onto the three-dimensional structure of the mouse catalytic subunit with a peptide inhibitor. Mutations defective in the recognition of both the regulatory subunit and the peptide substrate Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) mapped to the peptide-binding site shared by all substrates and inhibitors of the catalytic subunit and functionally define the binding site for the autoinhibitor sequence in the hinge region of the regulatory subunit. Mutants defective only in the recognition of the regulatory subunit identified residues that comprise additional binding sites for the regulatory subunit. The majority of these residues are clustered on the surface of the catalytic subunit in a region flanking the distal portion of the autoinhibitor/peptide-binding site. The simultaneous substitution of Lys233, Asp237, Lys257, and Lys261 in this region caused a 260-fold decrease in affinity for the regulatory subunit, whereas the catalytic efficiency toward Kemptide decreased by only 1.8-fold. The substitution of autophosphorylated Thr241, also in this region, and the 3 residues interacting with the phosphate also caused an unregulated phenotype.

Amino Acid Sequence↗

PubMed1992

cAMP-dependent protein kinase and the protein kinase family.

The structure of the catalytic subunit of cAMP-dependent protein kinase, the first protein kinase structure to be solved, is reviewed. The general architecture of the enzyme is described as well as the active site regions associated with substrate binding and catalysis. In particular, the unique features of the protein kinase nucleotide fold are outlined. While the catalytic subunit is one of the simplest of the protein kinases, it nevertheless serves as a structural framework for the catalytic core of the entire protein kinase family which now includes over 200 important regulatory enzymes. The essential and conserved features of this core are summarized, and a preliminary model of myosin light-chain kinase, based on the structure of the catalytic subunit, is also discussed.

Amino Acid Sequence↗

PubMed1992

Structural framework for the protein kinase family.

In this review, we have summarized the general structural features of the catalytic subunit of cAMP-dependent protein kinase, emphasizing those features that will very likely be conserved in all members of the protein kinase family. The overall secondary structure of the catalytic core will probably be conserved throughout the catalytic core, as will the active site regions associated with MgATP binding and catalysis. The mechanisms for activation and the role of protein phosphorylation are unique for each kinase. The structure of the catalytic subunit now provides a general framework for modeling other protein kinases. Although this is no substitute for a crystal structure for each protein kinase, this one structure, nevertheless, does provide major insights to the molecular organization of each of these enzymes.

Adenosine Triphosphate↗