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Kinetic studies of the phosphoenzyme intermediates of site-specific mutants were used to examine the role of Gly233in the reaction mechanism of the sarcoplasmic reticulum Ca2+-ATPase. When this glycine residue, which is highly conserved among cation-transporting ATPases, was replaced by valine, arginine, or glutamic acid, a complete loss of the ability to pump Ca2+was observed. The mutant enzymes were able to form an ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP in the presence of Ca2+, but this intermediate decayed to the ADP-insensitive form (E2P) very slowly, relative to the wild-type enzyme. The mutant phosphoenzyme intermediate remained ADP-sensitive, even when phosphorylation from ATP was performed under conditions which permitted accumulation of the ADP-insensitive phosphoenzyme intermediate in the wild type. The mutants were also defective in their ability to form the ADP-insensitive phosphoenzyme intermediate by phosphorylation from inorganic phosphate. In addition, they displayed a higher affinity for Ca2+and a lower cooperativity in Ca2+binding than did the wild-type enzyme, as measured through the phosphorylation reaction with ATP. These findings can be rationalized either in terms of a parallel shift of E1to E2and E1P to E2P conformational equilibria toward the E1and E1P forms, respectively, or in terms of destabilization of the phosphoryl-protein interaction in the E2P form. The roles of 7 other residues located in the vicinity of Gly233were also examined by mutation. Although the side chains of these residues are potential Ca2+ligands, their replacement did not affect the Ca2+affinity of the enzyme, suggesting the lack of a role of this region of the peptide in formation of Ca2+-binding sites.

Tissue contents of the sarcoplasmic-reticulum Ca2+-ATPase (Ca2+ +Mg2+-dependent ATPase), of calsequestrin and of parvalbumin were immunochemically quantified in homogenates of fast- and slow-twitch muscles of embryonic, maturing and adult rabbits. Unlike parvalbumin, Ca2+-ATPase and calsequestrin were expressed in embryonic muscles. Presumptive fast-twitch muscles displayed higher contents of these two proteins than did presumptive slow-twitch muscles. Calsequestrin steeply increased before birth and reached adult values in the two muscle types 4 days after birth. The main increase in Ca2+-ATPase occurred during the first 2 weeks after birth. Denervation of postnatal fast- and slow-twitch muscles decreased calsequestrin to amounts typical of embryonic muscle and suppressed further increases of Ca2+-ATPase. Denervation caused slight decreases in Ca2+-ATPase in adult fast-twitch, but not in slow-twitch, muscles, whereas calsequestrin was greatly decreased in both. Chronic low-frequency stimulation induced a rapid decrease in parvalbumin in fast-twitch muscle, which was preceded by a drastic decrease in the amount of its polyadenylated RNA translatable in vitro. Tissue amounts of Ca2+-ATPase and calsequestrin were essentially unaltered up to periods of 52 days stimulation. These results indicate that in fast- and slow-twitch muscles different basal amounts of Ca2+-ATPase and calsequestrin are expressed independent of innervation, but that neuromuscular activity has a modulatory effect. Conversely, the expression of parvalbumin is greatly enhanced by phasic, and drastically decreased by tonic, motor-neuron activity.

The aim of this study was to investigate the effects of neural activity on the expression of fibre-type-specific patterns of metabolic enzymes at the levels of transcription and translation. For this purpose, changes in tissue amounts of citrate synthase (CS) and the H- and M-subunits of lactate dehydrogenase (LDH) were followed in fast-twitch rabbit muscles during low-frequency (10 Hz, 12 h/day) nerve stimulation. These stimulation-induced alterations were correlated with changes in tissue amounts of the total poly(A)+ (polyadenylated) RNA, poly(A)+ RNAs specifically translatable in vitro, yield of total ribosomes and distributions of monosomes and polysomes. The tissue contents of poly(A)+ RNAs translatable in vitro coding for CS and H- and M-LDH were quantified by immunoprecipitation of their translation products. Increases in total ribosome yields occurred after 4 days' stimulation, reaching a maximum between 14 and 21 days. Stimulation for only 1-2 days greatly increased the amount of monosomes. An increase in polysomes occurred before that in total ribosomes, suggesting that monosomes were integrated into polysomes. Total poly(A)+ RNA significantly increased in muscles stimulated for more than 6 days. A maximum increase of 2.5-fold was attained after 14-21 days. Chronic stimulation progressively induced the appearance of LDH isoenzymes containing the H-subunit, with a predominance of LDH-3. This shift corresponded to a slow decay of the M-subunit and a 2-fold steep increase in the H-subunit. These changes correlated with those of the respective poly(A)+ RNAs translatable in vitro, thus indicating that the re-arrangement of the LDH isoenzyme pattern is mainly due to qualitatively and quantitatively altered transcription. The increase in CS was biphasic and consisted of a moderate rise during the first 4 days of stimulation and a steep rise thereafter. The latter coincided with a steep increase in poly(A)+ RNA translatable in vitro coding for CS. In view of the early increase in translational capacity, it was concluded that the initial rise in CS resulted from selective post-transcriptional control and enhanced translation in vivo of existing mRNA, whereas its steep increase was due to enhanced transcription. These results indicate that the neurally regulated expression of phenotype-specific properties in muscle includes control of both transcription and translation.

1. Fibre types, Ca2+‐sequestering proteins (parvalbumin, sarcoplasmic reticulum Ca2+‐ATPase), enzyme activities of energy metabolism, and lactate dehydrogenase isozymes were studied in extensor digitorum longus (EDL) and soleus muscles of adult rats after denervation and direct stimulation for 45‐61 days. The stimulation resembled the firing patterns of normal motor units (Hennig & Lømo, 1985) in either soleus (20 Hz, high amount) or EDL (150 Hz, low and high amount). 2. Low‐ and high‐amount 150 Hz stimulation maintained essentially normal properties in the denervated EDL and induced many EDL‐like properties in the denervated soleus, e.g. pronounced increases in Ca2+‐ATPase and parvalbumin contents, a rise in the glycolytic enzyme activities, as well as a reversal of the lactate dehydrogenase H/M subunit ratio. The 150 Hz high‐amount stimulation was not as efficient as the 150 Hz low amount in maintaining the fast properties of denervated EDL with the exception of the enzymes of aerobic oxidative metabolism. High‐amount 20 Hz stimulation maintained essentially normal properties in the soleus but induced only moderate slow muscle characteristics in the EDL. 3. The maintenance of essentially normal properties by 'native' and the changes induced by 'foreign' stimulus patterns in the absence of the nerve indicate that evoked muscle activity plays a major role in controlling the phenotypic expression of muscle properties. However, the different responses of the denervated fast‐ and slow‐twitch muscles to identical stimulus patterns further suggest that rat EDL and soleus muscles contain intrinsically different muscle fibres.

p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases such as Cdc42 and Rac. Since these GTPases play key roles in regulating cell polarity, stress responses, and cell cycle progression, the ability of PAK to affect these processes has been examined. We previously showed that fission yeast pak1+ encodes an essential protein that affects mating and cell polarity. Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe. Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p contains an N-terminal pleckstrin homology domain in addition to a p21-binding domain and a protein kinase domain that are common to other members of the PAK family. Unlike pak1+, pak2(+) is not essential for vegetative growth or for mating in S. pombe. Overexpression of the wild-type pak2+ allele suppresses the lethal growth defect associated with deletion of pak1+, and this suppression requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as well as kinase activity. A substantial fraction of Pak2p is associated with membranous components, an association mediated both by the pleckstrin homology- and by the p21-binding domains. These results show that S. pombe encodes at least two pak genes with distinct functions and suggest that the membrane localization of Pak2p, directed by its interactions with membrane lipids and Cdc42p, is critical to its biological activity.

Ste20p from Saccharomyces cerevisiae is a member of the Ste20/p21-activated protein kinase family of protein kinases. The Ste20p kinase is post-translationally modified by phosphorylation in a cell cycle-dependent manner, as judged by the appearance of phosphatase-sensitive species with reduced mobility on SDS-polyacrylamide gel electrophoresis. This modification is maximal during S phase, and correlates with the accumulation of Ste20p fused to green fluorescent protein at the site of bud emergence. Overexpression of Cln2p, but not Clb2p or Clb5p, causes a quantitative shift of Ste20p to the reduced mobility form, and this shift is dependent on Cdc28p activity. The post-translational mobility shift can be generated in vitro by incubation of Ste20p with immunoprecipitated Cln2p kinase complexes, but not by immunoprecipitated Clb2p or Clb5p kinase complexes. Ste20p is therefore a substrate for the Cdc28p kinase, and undergoes a Cln2p-Cdc28p mediated mobility shift as cells initiate budding and DNA replication. In cells that express only the Cln2p G1 cyclin, minor overexpression of Ste20p causes aberrant morphology, suggesting a proper coordination of Ste20p and Cln-Cdc28p activity may be required for the control of cell shape.

The budding yeast Saccharomyces cerevisiae has two functionally redundant myosin-I isoforms encoded by the MYO3 and MYO5 genes. The function shared by these myosin proteins is required for proper yeast budding. Serine residue 357 in the head domain of Myo3p, conserved among myosin-I proteins including yeast Myo5p, was identified as a unique phosphorylation site for the serine/threonine protein kinase Ste20p and its closely related isoform Cla4p. These protein kinases share a function that is also essential for budding. Replacement of serine 357 with alanine disrupted the in vivo function of Myo3p, whereas this function was maintained by changing the serine residue to aspartate. This mutant version failed to compensate the growth defect of cells which lack both Ste20p and Cla4p, suggesting that myosin-I is not the only essential target of these protein kinases. Our results suggest that phosphorylation of the head domain by Ste20p-like protein kinases plays an essential role in the function of myosin-I in yeast cells.

The heavy chain of myosin-ID isolated from Dictyostelium was identified as an in vitro substrate for members of the Ste20p family of serine/threonine protein kinases which are thought to regulate conserved mitogen-activated protein kinase pathways. Yeast Ste20p and Cla4p and mammalian p21-activated protein kinase (PAK) phosphorylated the heavy chain to 0.5-0.6 mol of Pi/mol and stimulated the actin-dependent Mg2+-ATPase activity to an extent equivalent to that of the Ste20p-like myosin-I heavy chain kinase isolated from Dictyostelium. PAK purified from rat brain required GTPgammaS-Cdc42 to express full activity, whereas recombinant mouse mPAK3 fused to glutathione S-transferase and purified from bacteria, and Ste20p and Cla4p purified from yeast extracts were fully active without GTPgammaS-Cdc42. These results suggest, together with the high degree of structural and functional conservation of Ste20p family members and myosin-I isoforms, that myosin-I activation by Ste20p family protein kinases may contribute to the regulation of morphogenetic processes in organisms ranging from yeast to mammalian cells.

The 53-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum was purified by lentil lectin affinity chromatography and preparative polyacrylamide gel electrophoresis and partially sequenced. Polyclonal and monoclonal antibodies were raised against the 53-kDa glycoprotein and found to cross-react with the 160-kDa glycoprotein.

Nucleotides encoding glutamate, glutamine, aspartate, or asparagine residues within the stalk sector of the sarcoplasmic reticulum Ca2+-ATPase were altered by oligonucleotide-directed site-specific mutagenesis. The mutant cDNAs were expressed in COS-1 cells, and mutant Ca2+-ATPases were assayed for Ca2+transport function and phosphoenzyme formation. Multiple mutations introduced into stalks, 1, 2, and 3 resulted in partial loss of Ca2+transport function. In most cases, subsequent mutation of individual amino acids in the cluster had no effect on Ca2+transport activity. In one cluster, however, it was possible to assign the reduction in Ca2+transport activity to alterations of Asn111and Asn114. The mutant Asn114to alanine retained about 50% activity, whereas the change Asn111to alanine retained only 10% activity. None of the mutations affected phosphorylation of the enzyme by ATP in the presence of Ca2+or by inorganic phosphate in the absence of Ca2+. The combined experiments suggest that the reduced Ca2+uptake observed in the Asn111and Asn114mutants was not due to a defect in enzyme activation by Ca2+or in formation of the phosphorylated enzyme intermediate but rather to incompetent handling of the bound Ca2+following ATP utilization. These results demonstrate that the acidic and amidated residues within the stalk region do not constitute the high affinity Ca2+-binding sites whose occupancy is required for enzyme activation. They may, however, act to sequester cytoplasmic Ca2+and to channel it to domains that are involved in enzyme activation and cation translocation.

Parvalbumin was purified from rabbit fast skeletal muscle and used to raise antibodies in sheep. Subsequently, a sensitive ‘sandwich’ enzyme-linked immunoadsorbent assay permitted quantification of parvalbumin in homogenates of embryonic, maturing, innervated, denervated and chronically stimulated skeletal muscles of the rabbit. High concentrations of parvalbumin were detected in various adult fast-twitch muscles of the rabbit (700-1200 micrograms/g of muscle), whereas slow-twitch muscles contained negligible concentrations (3-5 micrograms/g of muscle). Parvalbumin was not detectable in embryonic-rabbit muscles (21, 25, 28 days of gestation), either presumptive fast- or slow-twitch. However, parvalbumin concentrations did increase during postnatal development in presumptive fast-twitch muscles. Thus the onset of parvalbumin synthesis appears to be correlated with the neonatal-to-adult transition of motor-neuron activity [Navarrete & Vrbová (1983) Dev. Brain Res. 8, 11-19]. The increase of parvalbumin in maturing, presumptive fast-twitch muscle was suppressed by denervation. In the adult rabbit, denervation of the tibialis anterior muscle caused a reduction of parvalbumin to a level normally found in slow-twitch muscles. In contrast, the already low levels of parvalbumin in maturing and adult slow-twitch soleus muscle were unaffected by denervation. Chronic low-frequency stimulation of adult fast-twitch muscle resulted in a rapid reduction of parvalbumin to a level normally found in slow-twitch muscle. These data support the hypothesis that the expression of parvalbumin is under positive control of fast-type motor-neuron activity.

Summary A sensitive enzyme-linked immunoadsorbant assay was developed to quantify Ca2+ -ATPase and calsequestrin from sarcoplasmic reticulum in human muscle biopsies. Tissue levels of Ca2+ -ATPase and calsequestrin averaged 51.5 ± 28.1 and 6.4 ± 1.8mg/g muscle protein, respectively, in control muscles (means ± SD, n=12). The high sensitivity and specificity of the antibodies make the assay a useful tool in the diagnosis of human neuromuscular disorders where defects in sarcoplasmic reticulum function may be expected. The assay was applied to muscle biopsies from patients with myotonia congenita and paramyotonia congenita Eulenburg. The calsequestrin concentration was normal in all patient muscles. The Ca2+ -ATPase content was also within the normal range but varied considerably with the percentage distribution of slowtwitch fibres. This indicates that the prolonged relaxation observed in the muscles of patients with these disorders is not caused by faulty expression of Ca2+-ATPase and calsequestrin.

Extracellular signal-regulated protein kinase (ERK, or mitogen-activated protein kinase [MAPK]) regulatory cascades in fungi turn on transcription factors that control developmental processes, stress responses, and cell wall integrity. CEK1 encodes a Candida albicans MAPK homolog (Cek1p), isolated by its ability to interfere with the Saccharomyces cerevisiae MAPK mating pathway. C. albicans cells with a deletion of the CEK1 gene are defective in shifting from a unicellular budding colonial growth mode to an agar-invasive hyphal growth mode when nutrients become limiting on solid medium with mannitol as a carbon source or on glucose when nitrogen is severely limited. The same phenotype is seen in C. albicans mutants in which the homologs (CST20, HST7, and CPH1) of the S. cerevisiae STE20, STE7, and STE12 genes are disrupted. In S. cerevisiae, the products of these genes function as part of a MAPK cascade required for mating and invasiveness of haploid cells and for pseudohyphal development of diploid cells. Epistasis studies revealed that the C. albicans CST20, HST7, CEK1, and CPH1 gene products lie in an equivalent, canonical, MAPK cascade. While Cek1p acts as part of the MAPK cascade involved in starvation-specific hyphal development, it may also play independent roles in C. albicans. In contrast to disruptions of the HST7 and CPH1 genes, disruption of the CEK1 gene adversely affects the growth of serum-induced mycelial colonies and attenuates virulence in a mouse model for systemic candidiasis.

The Ste20p protein kinase was immunopurified from yeast cells and analyzed in an in vitro assay system. Ste20p immune complexes exhibited autophosphorylating activity at serine and threonine residues and specifically phosphorylated a bacterially expressed glutathione S-transferase (GST) fusion of Ste11p (a mitogen-activated protein or extracellular signal-regulated kinase kinase (MEK) kinase homologue) at serine and threonine residues. In contrast, GST fusions either of Ste7p (a MEK homologue) or the beta-subunit of the mating response G-protein and immunoprecipitated Ste5p were not phosphorylated by the Ste20p immune complexes. Myelin basic protein was identified as an excellent in vitro substrate, whereas histone H1 was only poorly phosphorylated. Evidence was obtained that autophosphorylation might play a regulatory role for the in vitro kinase activity. The in vitro activity was found to be Ca(2+)-independent. Both the in vivo and in vitro activities were abolished by mutational changes of either the conserved lysine residue 649 within the ATP binding site or threonine 777 between the catalytic subdomains VII and VIII. Wild-type Ste20p and the catalytically inactive T777A mutant were identified as phosphoproteins in vivo. The phosphorylation occurred at serine and threonine residues independent of pheromone stimulation. Based on the genetically determined significance of Ste20p in pheromone signal transduction and on our in vitro studies, we propose the model that Ste20p represents a yeast MEK kinase kinase whose function is to link G-protein-coupled receptors through G beta gamma to a mitogen-activated protein kinase module.

Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH2-terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca2+. The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45Ca2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca2+ with the membrane. We propose that this lumenal Ca2+ binding glycoprotein of the sarcoplasmic reticulum be designated "sarcalumenin."