Biomedical subjects
Publications and source records attributed to J D Scott.
Signal transduction in the nervous system is heavily dependent on the three multifunctional serine/threonine protein kinases, PKA, PKC, and CaM-KII. Recent studies have furthered our understanding of how the multiple isoforms of these kinases and their subcellular localizations, regulatory properties, and substrate determinants are important for the specificity of kinase functions.
The type II cAMP-dependent protein kinase (PKA) is localized in certain cellular compartments through association with specific A-kinase anchoring proteins (AKAPs). A variety of blotting and electrophoresis techniques have been developed to study the protein-protein interactions that occur between the regulatory (R) subunit of PKA and AKAPs. These methods have also been used for a variety of purposes such as detecting calmodulin-binding proteins, comparing wild-type- and mutant-form binding affinities and estimating the molecular weight of multiprotein complexes.
The type II cAMP-dependent protein kinase is localized to specific subcellular environments through the binding of the regulatory subunit (RII) dimer to RII-anchoring proteins. Computer-aided analysis of secondary structure, performed on four RII-anchoring protein sequences (the microtubule-associated protein 2, P150, and two thyroid proteins Ht 21 and Ht 31), has identified common regions of approximately 14 residues which display high probabilities of forming amphipathic helices. The potential amphipathic helix region of Ht 31 (Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile) lies between residues 494 and 507. A bacterially expressed 318-amino acid fragment, Ht 31 (418-736), containing the amphipathic helix region, was able to bind RII alpha. Site-directed mutagenesis designed to disrupt the secondary structure in the putative binding helix reduced binding dramatically. Specifically, substitution of proline for Ala-498 significantly diminished RII alpha binding, and similar mutation of Ile-502 or Ile-507 abolished interaction. Mutation of Ala-522 to proline, which is located outside the predicted amphipathic helix region, had no effect on RII alpha binding. These data suggest that anchoring proteins interact with RII alpha via an amphipathic helix binding motif.
The major function of the regulatory (R) subunit of the cAMP-dependent protein kinase is to bind tightly to the catalytic (C) subunit to form an inactive holoenzyme in the absence of cAMP. The hinge region of the R subunit resembles the substrate recognition site for the C subunit and is known to be involved in the R.C subunit interaction. Two arginine residues in this region, Arg-92 and Arg-93, are suggested to be essential for holoenzyme formation. In this study, a mutant in which Arg-92 and Arg-93 of type II regulatory subunit (RII) were replaced with alanine was constructed. Formation of the holoenzyme from mutant RII and C subunits was analyzed by gel-filtration and cation-exchange chromatography. Mutant RII in its cAMP-free form formed a stable holoenzyme with the C subunit, which dissociated in the presence of cAMP. Interestingly, the holoenzyme formed from mutant RII and C subunits retained full enzymatic activity even in the absence of cAMP. Although mutant RII could no longer be phosphorylated by the C subunit, the rate of [3H]cAMP release from mutant RII.cAMP was increased by addition of the C subunit, indicating that C-induced cAMP release is not the result of the interaction of the C subunit with the hinge region. These results demonstrate that Arg-92 and Arg-93 are not essential for holoenzyme formation but are critical for inhibiting kinase activity in the holoenzyme probably by occupying the substrate binding site. The results suggest that, in addition to the hinge region, a second site on the RII subunit may interact with the C subunit in a cAMP-dependent manner.
The actions of several hormones and neurotransmitters evoke signal transduction pathways which rapidly elevate the cytosolic concentrations of the intracellular messengers, cAMP and cGMP. The cyclic-nucleotide dependent protein kinases, cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), are the major intracellular receptors of cAMP and cGMP. These enzymes become active upon binding respective cyclic nucleotides and modulate a diverse array of biochemical events through the phosphorylation of specific substrate proteins. The focus of this review is to describe the progress made in understanding the structure and function of both PKA and PKG.
In previous work, we found that nerve growth factor (NGF) induced expression of the mRNA transcript encoding the metalloproteinase transin/stromelysin in PC12 cells. Transin was found, moreover, to be a "late" gene product whose expression correlated with neurites extension. In this study, various aspects of the NGF intracellular signaling pathway in PC12 cells are investigated. We show that the protein kinase inhibitor staurosporine, but not various other kinase inhibitors, specifically blocked the NGF induction of transin. Preliminary characterization of this staurosporine-sensitive kinase suggest that it does not correspond to a tyrosine kinase, nor various serine kinases, and that it is involved both at the transcriptional and posttranscriptional levels of transin gene regulation. In contrast to these effects of staurosporine, various activators of protein kinases C and A augmented the NGF induction of transin. Similar effects of these kinase inhibitors and activators were also observed with the expression of various immediate-early genes that have been proposed to mediate the transcriptional effects of NGF, including c-fos and c-jun. These data suggest, therefore, that the NGF induction of transin mRNA expression involves multiple protein kinases acting at a number of postreceptor regulatory steps in the NGF signaling pathway.
The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of dimeric regulatory subunits (RII) to anchoring proteins. Cytoskeletal localization occurs through RII dimer interaction with the PKA substrate molecule microtubule-associated protein 2 (MAP2). RII alpha deletion mutants and RII alpha/endonexin chimeras retained MAP2 binding activity if they contained the first 79 residues of the molecule. Disruption of RII alpha dimerization always prevented MAP2 interaction because 1) RII delta 1-14 (an amino-terminal deletion mutant lacking residues 1-14) was unable to bind MAP2 or form dimers, and 2) a modified RII alpha monomer including residues 1-14 did not bind MAP2. Chimeric proteins containing the first 30 residues of RII alpha fused to endonexin II formed dimers but did not bind MAP2. This suggested other side-chains between residues 30-79 also participate in MAP2 interaction. Peptide studies indicate additional contact with MAP2 may occur through an acidic region (residues 68-82) close to the RII autoinhibitor domain. Therefore, anchored PKA holoenzyme topology may position the catalytic subunit and MAP2 as to allow its preferential phosphorylation upon kinase activation.
The cellular and subcellular distribution of a major cyclic AMP binding protein in the central nervous system, the neural-specific regulatory subunit of the type II cyclic AMP-dependent protein kinase (RII-B), was analyzed in rat brains with light and electron microscopic immunocytochemical methods. The distribution of the non-neural isoform of the regulatory subunit of the enzyme (RII-H) was also analyzed. It was found that RII-B immunoreactivity was predominantly localized to neurons whereas glial and endothelial cells were unlabeled. In the neurons the RII-B immunoreactivity occurred in the perikaryal cytoplasm and in the dendrites; there was no significant accumulation of immunoreaction product in nuclei, myelinated axons and axon terminals. Although immunoreactivity was never detected in axon terminals, it was characteristically associated with the postsynaptic densities and the surrounding non-synaptic sites in somata, dendrites and dendritic spines. The localization of RII-B antigenic sites did not show specificity to any type of neuron or synapse, but the amount of immunoreactivity varied. The distribution of RII-H immunoreactivity was similar to that of RII-B except that RII-H immunoreaction product was also observed in glial cells and occurred more frequently in myelinated axons. Our data confirm that RII-B is one of the major cyclic AMP binding proteins in neurons, and provide morphological support for the involvement of the type II cyclic AMP-dependent protein kinase in postsynaptic neural functions.
Several chemical delivery systems (CDS) were synthesized for the cholinesterase inhibitor 9-amino-1,2,3,4-tetrahydroacridine (THA). The derivatives prepared were substituted with a 1,4-dihydropyridine in equilibrium pyridinium salt redox system at the amino functionality. These compounds were synthesized by acylation of the 9 amino group of THA with nicotinic anhydride under forced conditions, followed by a selective N-alkylation of the pyridine ring and regioselective reduction of the resulting quaternary salts. Lipophilicity parameters indicated increased lipophilic indices for various CDS's compared to the THA. Oxidation studies showed that dihydronicotinamides readily converted to the quaternary salt, both chemically and enzymatically. The transport forms of THA were also shown not to interact with acetylcholinesterase in vivo. In vivo distribution studies in the rat indicated that high and sustained levels of the pyridinium quaternary ion derivative were present in the central nervous system (CNS). In addition, THA was produced in the CNS from the quaternary salt precursor in low concentrations, indicating a slow but sustained release. The CDS for THA were found to be less acutely toxic than THA.
Superior peripheral iridectomy has been performed in 40 patients who have undergone silicone oil surgery for retinal detachment and who are aphakic. Only two developed raised intraocular pressure due to pupil block by liquid silicone and in each case the iridectomies seemed to be closed by proliferative membrane rather than the silicone meniscus. This type of iridectomy is particularly suitable in patients requiring intracapsular cataract extraction after previous vitrectomy and silicone oil surgery.
Cyclic AMP (cAMP) and cAMP-dependent protein kinases (PKAs) are believed to be involved in the regulation of essential spermatozoal functions, such as motility, epididymal maturation, capacitation, and the acrosome reaction. In this study, we document the presence of significant mRNA levels for 5 different PKA subunits (RI alpha, RI beta, RII alpha, RII beta, and C alpha) in germ cells and demonstrate differential expression patterns for these subunits during spermatogenesis. Messenger RNAs for RI (RI alpha and RI beta) and C alpha appear to be induced at premeiotic germ cell stages, whereas mRNAs for RII (RII alpha and RII beta) are first expressed at haploid stages. The individual PKA subunits may convey specific functions in developing germ cells and mature sperm. The present study, furthermore, demonstrates the presence of unique smaller-sized mRNAs in germ cells compared with somatic cells. Specific, truncated forms of RI alpha, RII alpha, RII beta, and C alpha mRNAs appear to be selected in the germ cells. Our data suggest this to be due to the use of alternative polyadenylation site signals. The selection of shorter mRNA species, with higher stability, may be essential for the delayed translation observed in spermatids. This may ensure certain levels of mRNA for translation at late spermatid stages, after cessation of transcription.
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Phosphorylations catalyzed by cAMP-dependent protein kinase are essential for sperm motility, and type II cAMP-dependent protein kinase in mature sperm has been shown to be firmly bound to the flagellum via the regulatory subunit, RII. The present study documents high-levelled expression of a human, testis-specific RII alpha mRNA (2.0 kb) analogous to the rat mRNA which is induced in haploid germ cells [(1988) FEBS Lett. 229, 391-394]. We report the molecular cloning of a full-length human cDNA corresponding to this unique testis mRNA, and the presence of an alternative amino-terminal region (amino acids 45-75) of the predicted RII alpha protein (404 amino acids) compared with the previously published mouse and rat sequences. However, this alternate region is also shown to be present in RII alpha mRNA (7.0 kb) of human somatic cells. Our data indicate the divergent amino-terminal sequence to be due to species differences, suggesting an active evolutionary pressure on this particular region, which could be involved in subcellular attachment of RII alpha and thereby localization of kinase activity to certain targets within the cell.
The levels of the regulatory (RI and RII) and catalytic (C) components of cAMP-dependent protein kinase and of their messages were studied during the first 36 h of liver regeneration after 70% hepatectomy. Both RI alpha mRNA and RII alpha mRNA started to increase 4 h after the resection, reaching peak levels after 9 h. RI mRNA decreased abruptly 9-12 h after resection, whereas RII mRNA stayed elevated. C alpha mRNA was rather constant during the period of study. In accordance with the mRNA data the level of C was constant while RI and RII increased during the prereplicative phase of liver regeneration. RI increased rapidly when its message became elevated. RII, however, increased noticeably only 6-8 h after its mRNA had become elevated. The increased expression of R led to a disproportion between R and C that was most pronounced 14 h after resection, i.e. coinciding with the prereplicative cAMP burst. The increased R/C ratio at that time of regeneration diminished the concentration of active C subunit during the cAMP burst. In that way the otherwise inhibitory effect of high concentrations of active C on the DNA replication may have been decreased. The fractional saturation of RI and RII by endogenous cAMP fluctuated in parallel as a function of liver cAMP levels, although there was a tendency that RI was more highly saturated than RII at high concentrations of cAMP.
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A cDNA clone encoding skeletal muscle myosin light chain kinase (MLCK) was isolated from a rat skeletal muscle library using oligonucleotide probes. The total length of the rat skeletal muscle MLCK cDNA was 2823 base pairs with an open reading frame of 1830 base pairs. The deduced sequence of the 610-amino acid protein exhibited 96% amino acid identity to rabbit skeletal muscle MLCK in the carboxyl-terminal portion of the molecule, which contains the catalytic and the calmodulin-binding domains, and 58% identity in the amino-terminal region. Analysis of total rat mRNA revealed a single mRNA species of 3.4 kilobases that was unique to skeletal muscle. Further analysis of skeletal muscle tissue using fast-twitch glycolytic, fast-twitch oxidative glycolytic, and slow-twitch oxidative fibers isolated from rat leg revealed that the mRNA level for MLCK varied among the three fiber types. The results of kinase assays performed on the fibers showed that MLCK activity levels paralleled the MLCK mRNA levels found in each of the three types of skeletal muscle fibers studied. Fast-twitch oxidative glycolytic (gastrocnemius red) and slow-twitch oxidative (soleus) exhibited 60 and 13%, respectively, of the enzymatic activity present in fast-twitch glycolytic (gastrocnemius white) fibers.
Cyclic AMP (cAMP) and its action by way of cAMP-dependent protein kinase is important for sperm motility. Previous studies on germ cells have demonstrated a selective decrease in the amount of type I cAMP-dependent protein kinase during spermatid development, and that type II was the major form present in elongating spermatids and in mature sperm. This would indicate activation of a gene in haploid germ cells, encoding a regulatory subunit of type II protein kinase. However, haploid expression of such a gene has so far not been shown. In the present study we demonstrate high-levelled expression of a unique mRNA species for a specific regulatory subunit of type II cAMP-dependent protein kinase at late stages of spermatogenesis, i.e. during spermatid elongation.