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S S Taylor

Publications and source records attributed to S S Taylor.

At least 73 records · Page 4Linked to original sources
PubMed1997

Synergistic binding of nucleotides and inhibitors to cAMP-dependent protein kinase examined by acrylodan fluorescence spectroscopy.

We have engineered an acrylodan-modified derivative of the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK) whose fluorescence emission signal has allowed the synergistic binding between nucleotides and physiological inhibitors of cAPK to be examined (Whitehouse, S., and Walsh, D. A. (1983) J. Biol. Chem. 258, 3682-3692). In the presence of the regulatory subunit, RI, the affinity of cAPK for adenosine, ADP, AMPPNP (adenosine 5'-(beta, gamma-imino)triphosphate), or ATP was 5-, 50-, 120-, and 15,000-fold enhanced, while in the presence of the heat-stable inhibitor protein of cAPK (PKI), there was a 3-, 20-, 33-, and 2000-fold enhancement in the binding of these nucleotides, respectively. A short inhibitor peptide, PKI-(14-22), enhanced the binding of ADP to the same degree as did full-length PKI (20-fold) but, in contrast, did not significantly enhance the binding of ATP or AMPPNP. The full binding synergism between PKI and either ATP (2000-fold) or AMPPNP (33-fold) to cAPK could, however, be mimicked by a longer peptide, PKI-(5-24), suggesting that the PKI NH2 terminus (residues 5-13) is most likely critical. Since this region is remote from the ATP gamma-phosphate, the binding synergism must arise through an extended network communication mechanism between the PKI NH2 terminus and the ATP binding site.

2-Naphthylamine↗
PubMed1997

Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface.

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI). The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.

Catalysis↗
PubMed1997

Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP-dependent protein kinase.

Two classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP-dependent protein kinase (cAPK), the heat-stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits. Basic sites on C, previously identified as important for R/C interaction in yeast TPK1 and corresponding to Lys213, Lys217, and Lys189 in murine C alpha, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI. rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI. This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994). Thus, the C-subunit employs two distinct electrostatic surfaces to achieve high-affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft. Unlike TPK1, mutation of Lys189 had no effect. The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C. Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations. rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C. The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.

Alanine↗
PubMed1997

Protein kinase inhibition: natural and synthetic variations on a theme.

How a protein kinase is turned off is as critical for its physiological function as is its catalytic activity. Examination of solved crystal structures representing different protein kinase subfamilies reveals a variety of strategies that are utilized by nature to lock protein kinases into inactive conformations. Pseudosubstrate and adenine mimetic mechanisms as well as complementarity to surfaces other than the active site are effective. Although most synthetic or natural product inhibitors target the active site, specifically the ATP binding site, a remarkably high degree of specificity can be achieved which is due to the extended surface of the protein that these inhibitors occupy. Although targeting of the ATP binding site is proving to be very successful, there is also wide latitude for designing inhibitors that target other surfaces of the kinases.

Enzyme Inhibitors↗
PubMed1997

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase in Escherichia coli.

When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is expressed in Escherichia coli, it is autophosphorylated at four sites, Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338 and Thr197, are also found in the mammalian enzyme. To understand the functional importance of these phosphorylation sites, each was replaced with Ala, Glu or Asp. The expression, solubility and phosphorylation state of each mutant protein was characterized by immunoprecipitation following in vivo labeling with 32Pi. When possible, isoforms were resolved and kinetic properties were measured. The two stable phosphorylation sites in the mammalian enzyme, Ser338 and Thr197, were shown to play different roles. Ser338, which stabilizes a turn near the C-terminus, is important for stability. Both rC(S338A) and rC(S338E) were very labile; however, the kinetic properties of rC(S338E) were similar to the wild-type catalytic subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to the surface of the small lobe. Thr197 is in the activation loop near the cleft interface. Mutagenesis of T197 caused a significant loss of catalytic activity with increases in Kms for both peptide and MgATP, as well as a small decrease in k(cat) indicating that this phosphate is important for the correct orientation of catalytic residues at the active site. Replacement of Ser139, positioned at the beginning of the E-helix, with Ala had no effect on the kinetic parameters, stability or phosphorylation at the remaining sites. In contrast, mutation of Ser10, located at the beginning of the A-helix, produced mostly insoluble, inactive, unphosphorylated protein, suggesting that this region, though far removed from the active site, is structurally important at least for the expression of soluble phosphoprotein in E.coli. Since the mutation of active site residues as well as deletion mutants generate underphosphorylated proteins, these phosphorylations in E.coli all result from autophosphorylation.

Adenosine Triphosphate↗
PubMed1997

Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D H NMR and molecular dynamics.

Peptides derived from the inhibitor of cAMP-dependent protein kinase. PKI, have been studied by 2D 1H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala20-Ser21]PKI(5-24), and a phosphorylated form of the latter [Ala20-Ser21P]PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI(5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly14-Gly17 turn was apparent in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp9 and Arg15. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2 degrees C) shows the occurrence of pH-dependent contacts with Phe10. We conclude that the binding of short inhibitors, such as PKI(5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala20-Ser21]PKI(5-24) and the product [Ala20-Ser21P]PKI(5-24), give very similar structures with local rearrangements involving some of the side chains.

Amino Acid Sequence↗
PubMed1996

A method for linking yeast artificial chromosomes.

A method for linking any standard yeast artificial chromosomes (YAC) is described. YACs are introduced into the same cell and joined by mitotic recombination between the vector arms and the homologous sequence in a linking vector; several YACs can be recombined sequentially. The linking vectors also contain the beta-galactosidase gene as an expression reporter in mammalian cells.

Cell Line↗
PubMed1996

Specificity of LIM domain interactions with receptor tyrosine kinases.

LIM domains, Cys-rich motifs containing approximately 50 amino acids found in a variety of proteins, are proposed to direct protein*protein interactions. To identify structural targets recognized by LIM domains, we have utilized random peptide library selection, the yeast two-hybrid system, and glutathione S-transferase fusions. Enigma contains three LIM domains within its carboxyl terminus and LIM3 of Enigma specifically recognizes active but not mutant endocytic codes of the insulin receptor (InsR) (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090). Interaction of two random peptide libraries with glutathione S-transferase-LIM3 of Enigma indicated specific binding to Gly-Pro-Hyd-Gly-Pro-Hyd-Tyr-Ala corresponding to the major endocytic code of InsR. Peptide competition demonstrated that both Pro and Tyr residues were required for specific interaction of InsR with Enigma. In contrast to LIM3 of Enigma binding to InsR, LIM2 of Enigma associated specifically with the receptor tyrosine kinase, Ret. Ret was specific for LIM2 of Enigma and did not bind other LIM domains tested. Mutational analysis indicated that the residues responsible for binding to Enigma were localized to the carboxyl-terminal 61 amino acids of Ret. A peptide corresponding to the carboxyl-terminal 20 amino acids of Ret dissociated Enigma and Ret complexes, while a mutant that changed Asn-Lys-Leu-Tyr in the peptide to Ala-Lys-Leu-Ala or a peptide corresponding to exon16 of InsR failed to disrupt the complexes, indicating the Asn-Lys-Leu-Tyr sequence of Ret is essential to the recognition motif for LIM2 of Enigma. We conclude that LIM domains of Enigma recognize tyrosine-containing motifs with specificity residing in both the LIM domains and in the target structures.

3T3 Cells↗
PubMed1996

Mitogenic signaling by Ret/ptc2 requires association with enigma via a LIM domain.

The ret/ptc2 papillary thyroid cancer oncogene, an oncogenic form of the c-Ret receptor tyrosine kinase, is the product of a somatic crossover event fusing the dimerization domain of the type Ialpha regulatory subunit of cyclic AMP-dependent protein kinase (RI) with the tyrosine kinase domain of c-Ret. Mitogenic activity of Ret/ptc2 required dimerization via the N terminus of RI and a tyrosine residue located C-terminal to the kinase core of Ret, Tyr-586 (Durick, K., Yao, V. J., Borrello, M. G., Bongarzone, I., Pierotti, M. A. and Taylor, S. S. (1995) J. Biol. Chem. 270, 24642-24645). Using the yeast two-hybrid system, Ret/ptc2 binding proteins were identified, and the sites of interaction with Ret/ptc2 were mapped. The SH2 domains of phospholipase Cgamma and Grb10 were both identified, and binding depended on phosphorylation of Tyr-539 and Tyr-429, respectively. These interactions, however, were not required for mitogenic signaling. The second of the three LIM domains in Enigma (Wu, R. Y., and Gill, G. N. (1994) J. Biol. Chem. 269, 25085-25090) was also identified as a Ret/ptc2 binding domain. Enigma, a 455-residue protein, was discovered based on its interaction with the insulin receptor through the C-terminal LIM domain. Although the association with Enigma required Tyr-586 of Ret/ptc2, the interaction was phosphorylation-independent. In contrast to the SH2 interactions, disruption of the interaction with Enigma abolished Ret/ptc2 mitogenic signaling, suggesting that LIM domain recognition of an unphosphorylated tyrosine-based motif is required for Ret signal transduction.

3T3 Cells↗
PubMed1996

Functional malleability of the carboxyl-terminal tail in protein kinase A.

The catalytic (C) subunit of protein kinase A (PKA) is regarded as a framework for the protein kinase family. Its sequence is composed of a conserved core (residues 40 300) between two segments at the amino and carboxyl termini of the protein. Since the various protein kinases differ in their specificity, it seems reasonable to assume that these nonhomologous segments may be involved in endowing each kinase with its individual specificity. Here we present data to show that the cluster of acidic amino acids (328DDYEEEE334) at the carboxyl-terminal "tail" of the C subunit, specifically Tyr330, contributes to its substrate recognition. This is based on three complementary lines of evidence: (i) on a conformation-sensitive cleavage of the C subunit by a kinase-splitting membranal proteinase that specifically recognizes this cluster, to demonstrate the occurrence in solution of "open" (cleavable) and "closed" (noncleavable) conformations of the C subunit; (ii) on analysis of the three-dimensional structures of the open and closed conformations of the C subunit, showing an approximately 7-A movement of the phenolic hydroxyl of Tyr330 to reach (in the closed conformation) an approximately 3-A distance from the nitrogen atoms of the Arg residue at position p-3 of the PKA consensus sequence; and (iii) on single-site mutations of the C subunit (e.g. Y330A) that show a significant contribution of Tyr330 to the Km of PKA for its substrates/inhibitors and to its catalytic efficacy (Vmax/Km).

Amino Acid Sequence↗
PubMed1996

Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase.

cAMP-dependent protein kinase (cAPK) is a heterotetramer containing two regulatory (R) and two catalytic (C) subunits. Each R-subunit contains two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domains. Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activation and to delineate the unique roles that each cAMP-binding domain plays. Two mutations were introduced by replacing the essential Arg in each cAMP-binding site with Lys (R209K in Site A and R333K in Site B). Also, the double mutant (R209/333K) was constructed. Analysis of cAMP binding and dissociation and the apparent constants for holoenzyme activation and R- and C-subunit interaction, measured by analytical gel filtration and surface plasmon resonance, established the following: (1) For rR(R209K), occupancy of Site B is not sufficient to activate the holoenzyme; the low affinity Site A must also be occupied. In rR(R333K), Site A retains its high affinity for cAMP, but Site A cannot bind until the low affinity Site B is occupied. Thus, both mutants, for different reasons, have similar Ka's for activation that are approximately 20-fold higher than that of the wild-type holoenzyme. The double mutant with two defective sites is no worse than either single mutant. (2) Kinetic analysis of cAMP binding showed that the mutation in Site A or B abolishes high affinity cAMP binding to that site and slightly weakens the affinity of the adjacent site for cAMP. (3) In the presence of MgATP, both mutants rapidly form a stable holoenzyme even in the presence of cAMP in contrast to the wild-type R where holoenzyme forms slowly in vitro and requires dialysis. Regarding the mechanism of activation based on these and other mutants and from kinetic data, the following conclusions are reached: Site A provides the major contact site with the C-subunit; Site B is not essential for holoenzyme formation. Occupancy of Site A by cAMP mediates dissociation of the C-subunit. Site A is inaccessible to cAMP in the full length holoenzyme, while Site B is fully accessible. Access of cAMP to Site A is mediated by Site B. Thus Site B not only helps to shield Site A, it also provides the specific signal that "opens up" Site A. Finally, a nonfunctional Site A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.

Amino Acid Sequence↗
PubMed1996

Catalytic subunit of cAMP-dependent protein kinase: electrostatic features and peptide recognition.

The electrostatic field was calculated for the mammalian cAMP-dependent protein kinase (PKA) catalytic subunit (C-subunit) complexed with a 20-residue peptide from a heat stable protein kinase inhibitor (PKI: 5-24). The electrostatic field was also calculated for the C-subunit complexed with a modeled heptapeptide substrate that has been used extensively in structure/function studies for the C-subunit. Perturbations in the electrostatic free energy were calculated when single ionizable active site residues were mutated to alanine. These perturbations in electrostatic free energy were correlated to changes in the binding energy measured in a charge-to-alanine scan of the homologous yeast C-subunit by M. J. Zoller and C. S. Gibbs [(1991) Journal of Biological Chemistry, Vol. 266, pp. 8923-8931; C. S. Gibbs and M. J. Zoller (1991) Biochemistry, Vol. 30, p. 22]. This analysis indicated that the substrate binding parameters primarily depend on electrostatic interactions between a substrate or inhibitor and the C-subunit. Amino acid replacements that led to large perturbations in the electrostatic field are listed in the text. pKa shifts were also calculated for the substrate's phosphate accepting atom, the serine hydroxyl oxygen, when the active site ionizable residues were changed to structurally similar uncharged amino acids. The theoretical mutation of three active site residues caused large shifts in this parameter: E91Q, D166N, and D184N. The calculated pKa shifts for these mutants indicate that the rate of phosphotransfer should be markedly reduced in these cases. This prediction has been experimentally confirmed for the D166N mutant. The correlation between calculated electrostatic free energy changes and measured binding energy, and pKa shifts with phosphotransfer for C-subunit mutants were within experimental error of the measurements. The calculations of electrostatic energy and delta pKa have identified previously unconsidered active site residues in the mammalian C-subunit that contribute to binding energy and phosphotransfer.

Animals↗
PubMed1996

Examination of an active-site electrostatic node in the cAMP-dependent protein kinase catalytic subunit.

The active site of the cAMP-dependent protein kinase catalytic subunit harbors a cluster of acidic residues-Asp 127, Glu 170, Glu 203, Glu 230, and Asp 241-that are not conserved throughout the protein kinase family. Based on crystal structures of the catalytic subunit, these amino acids are removed from the site of phosphoryl transfer and are implicated in substrate recognition. Glu 230, the most buried of these acidic residues, was mutated to Ala (rC[E230A]) and Gln (rC[E230Q]) and overexpressed in Escherichia coli. In contrast to the mostly insoluble and destabilized rC[E230A], rC[E230Q] is largely soluble, purifies like wild-type enzyme, and displays wild-type-like thermal stability. The mutation in rC[E230Q] causes an order of magnitude decrease in the affinity for a heptapeptide substrate, Kemptide. In addition, two independent kinetic techniques were used to dissect phosphoryl transfer and product release steps in the reaction pathway. Viscosometric and pre-steady-state quench-flow analyses revealed that the phosphoryl transfer rate constant decreases by an order of magnitude, whereas the product release rate constant remains unperturbed. Electrostatic alterations in the rC[E230Q] active site were assessed using modeling techniques that provide molecular interpretations for the substrate affinity and phosphoryl transfer rate decreases observed experimentally. These observations indicate that subsite recognition elements in the catalytic subunit make electrostatic contributions that are important not only for peptide affinity, but also for catalysis. Protein kinases may, therefore, discriminate substrates by not only binding them tightly, but also by only turning over ones that complement the electrostatic character of the active site.

Animals↗
PubMed1996

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells.

Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes. In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures. These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection. These observations suggest that, despite the presence of centromeric sequences, the structures were not segregating efficiently and thus were not forming artificial chromosomes. However, extrachromosomal structures containing alphoid DNA appeared cytogenetically smaller than those lacking it, as long as yeast DNA was also absent. This suggests that alphoid DNA can generate the condensed chromatin structure at the centromere.

Animals↗
PubMed1995

Kinetic analysis of cAMP-dependent protein kinase: mutations at histidine 87 affect peptide binding and pH dependence.

Mutation of His87 in the catalytic (C-) subunit of the cAMP-dependent protein kinase (cAPK) led to changes in the kinetic properties of this enzyme. The C-subunit is a bilobal structure, with catalysis occurring in the cleft between the two lobes. His87 lies at the edge of the cleft, making an interaction with phosphothreonine, 197. This is the only direct electrostatic or hydrogen-bonding interaction between the small and large lobes. Solvent viscosity studies of the His87Ala mutant of the C-subunit (rC[H87A]) revealed that binding of two peptides, LRRASLG and LRRASLG-NH2, was impaired relative to that of the wild-type C-subunit. Consistent with this, the Ki's for two inhibitor peptides, LRRAALG and LRRAALG-NH2, were 4 and 1.4 mM, respectively, 5- and 7-fold higher than the Ki's of the respective peptides for wild-type protein. Kinetic constants for three octapeptide substrates that differed only at the P+2 position suggested a direct interaction of His87 with residues at this site. The kcat for rC[H87A] was 2-3-fold higher than kcat for the wild-type enzyme, indicating an effect of the mutation on the rate-limiting step, product release. The pH dependence of kinetic parameters for rC[H87A] was also measured. A single pKa of 6.5 was observed in kcat/Kpeptide as compared to the two pKa's of 6.5 and 8.5 for the wild-type enzyme. These changes suggest a role for His87 in substrate recognition and in stabilization of the catalytically competent conformation of the enzyme.

Amino Acid Sequence↗
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↗