cAMP-dependent Protein Phosphorylation Research Articles

Dolichol Phosphate Mannose Synthase: A Glycosyltransferase with Unity in Molecular Diversities

N‐glycans provide structural and functional stability to asparagine‐linked (N‐linked) glycoprotein, and add flexibility. Glycan biosynthesis is elaborative, multi‐compartmental and involves many glycosyltransferases. Failure to assemble N‐glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N‐glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol‐linked tetra‐decasaccharide (Glc3Man9GlcNAc2‐PP‐Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role. DPMS is also essential for GPI anchor biosynthesis as well as for O‐ and C‐mannosylation of proteins in yeast and in mammalian cells. DPMS has been purified from several sources and its gene has been cloned from 39 species (e.g., from protozoan parasite to human). It is an inverting GT‐A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org). The sequence alignment detects the presence of a metal binding DAD signature in DPMS from all 39 species but finds cAMP‐dependent protein phosphorylation motif (PKA motif) in only 38 species. DPMS also has hydrophobic region(s). Hydropathy analysis of amino acid sequences from bovine, human, S. crevisiae and A. thaliana DPMS show PKA motif is present between the hydrophobic domains. The location of PKA motif as well as the hydrophobic domain(s) in the DPMS sequence vary from species to species. For example, the domain(s) could be located at the center or more towards the C‐terminus. Irrespective of their catalytic similarity, the DNA sequence, the amino acid identity, and the lack of a stretch of hydrophobic amino acid residues at the C‐terminus, DPMS has been classified as Type I and Type II enzyme. DPMS belonging to Type II sub‐class has been considered to be a multimeric protein requiring the DPM2 and DPM3 to anchor down the DPM1 to the ER membrane. Our results indicate that the recombinant DPMS from bovine adrenal medullary capillary endothelial cells can be expressed successfully in E. coli as an active enzyme. Furthermore, DPMS is regulated by extracellular signaling and microenvironment. So, we conclude that it is the hydrophobic domain that anchor down DPMS to the ER membrane.Support or Funding InformationSupported in part by the grant NIH/NIMHD G12MD007583 (KB)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dolichol phosphate mannose synthase: a Glycosyltransferase with Unity in molecular diversities.

N-glycans provide structural and functional stability to asparagine-linked (N-linked) glycoproteins, and add flexibility. Glycan biosynthesis is elaborative, multi-compartmental and involves many glycosyltransferases. Failure to assemble N-glycans leads to phenotypic changes developing infection, cancer, congenital disorders of glycosylation (CDGs) among others. Biosynthesis of N-glycans begins at the endoplasmic reticulum (ER) with the assembly of dolichol-linked tetra-decasaccharide (Glc3Man9GlcNAc2-PP-Dol) where dolichol phosphate mannose synthase (DPMS) plays a central role. DPMS is also essential for GPI anchor biosynthesis as well as for O- and C-mannosylation of proteins in yeast and in mammalian cells. DPMS has been purified from several sources and its gene has been cloned from 39 species (e.g., from protozoan parasite to human). It is an inverting GT-A folded enzyme and classified as GT2 by CAZy (carbohydrate active enZyme; http://www.cazy.org ). The sequence alignment detects the presence of a metal binding DAD signature in DPMS from all 39 species but finds cAMP-dependent protein phosphorylation motif (PKA motif) in only 38 species. DPMS also has hydrophobic region(s). Hydropathy analysis of amino acid sequences from bovine, human, S. crevisiae and A. thaliana DPMS show PKA motif is present between the hydrophobic domains. The location of PKA motif as well as the hydrophobic domain(s) in the DPMS sequence vary from species to species. For example, the domain(s) could be located at the center or more towards the C-terminus. Irrespective of their catalytic similarity, the DNA sequence, the amino acid identity, and the lack of a stretch of hydrophobic amino acid residues at the C-terminus, DPMS is still classified as Type I and Type II enzyme. Because of an apparent bio-sensing ability, extracellular signaling and microenvironment regulate DPMS catalytic activity. In this review, we highlight some important features and the molecular diversities of DPMS.

Acute Hypoglycemia in Healthy Humans Impairs Insulin-Stimulated Glucose Uptake and Glycogen Synthase in Skeletal Muscle: A Randomized Clinical Study.

Hypoglycemia is the leading limiting factor in glycemic management of insulin-treated diabetes. Skeletal muscle is the predominant site of insulin-mediated glucose disposal. Our study used a crossover design to test to what extent insulin-induced hypoglycemia affects glucose uptake in skeletal muscle and whether hypoglycemia counterregulation modulates insulin and catecholamine signaling and glycogen synthase activity in skeletal muscle. Nine healthy volunteers were examined on three randomized study days: 1) hyperinsulinemic hypoglycemia (bolus insulin), 2) hyperinsulinemic euglycemia (bolus insulin and glucose infusion), and 3) saline control with skeletal muscle biopsies taken just before, 30 min after, and 75 min after insulin/saline injection. During hypoglycemia, glucose levels reached a nadir of ∼2.0 mmol/L, and epinephrine rose to ∼900 pg/mL. Hypoglycemia impaired insulin-stimulated glucose disposal and glucose clearance in skeletal muscle, whereas insulin signaling in glucose transport was unaffected by hypoglycemia. Insulin-stimulated glycogen synthase activity was completely ablated during hyperinsulinemic hypoglycemia, and catecholamine signaling via cAMP-dependent protein kinase and phosphorylation of inhibiting sites on glycogen synthase all increased.

CFTR: a missing link between exocrine and endocrine pancreas?

CFTR: a missing link between exocrine and endocrine pancreas?

Distinct roles of soluble and transmembrane adenylyl cyclases in the regulation of flagellar motility in Ciona sperm.

Adenylyl cyclase (AC) is a key enzyme that synthesizes cyclic AMP (cAMP) at the onset of the signaling pathway to activate sperm motility. Here, we showed that both transmembrane AC (tmAC) and soluble AC (sAC) are distinctly involved in the regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC inhibitor blocked both cAMP synthesis and the activation of sperm motility induced by the egg factor sperm activating and attracting factor (SAAF), as well as those induced by theophylline, an inhibitor of phoshodiesterase. It also significantly inhibited cAMP-dependent phosphorylation of a set of proteins at motility activation. On the other hand, a sAC inhibitor does not affect on SAAF-induced transient increase of cAMP, motility activation or protein phosphorylation, but it reduced swimming velocity to half in theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming trajectory with smaller diameter and significantly suppressed chemotaxis of sperm to SAAF. These results suggest that tmAC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat frequency and in the Ca2+-dependent chemotactic movement of sperm.

Cyclic AMP as a Regulator of the Phototransduction Cascade

Until recently, cAMP has been believed to play an important role in the organization of circadian rhythms in the vertebrate eye but not to be directly involved in controlling the phototransduction cascade in photoreceptors. However, given that cAMP levels in photoreceptor cells vary significantly during the day and that cAMP metabolism in photoreceptor cells may itself be light-dependent, it is logical to suggest that cAMP-dependent protein phosphorylation may be a mechanism for tuning phototransduction processes in accordance with ongoing illumination. The current review addresses contemporary data on the structure of the circadian pacemaker in the retina, the involvement of intraphotoreceptor cAMP in its organization, and the known intracellular targets on which cAMP has or may have regulatory influences. Our recent data indicate that changes in cAMP levels have regulatory influences on the photoreceptor cascade. This action may significantly widen the range of adaptation of photoreceptors to illumination conditions, increasing its sensitivity during the dark part of the day and decreasing its sensitivity in bright light.

Antiplatelet Effects of Caffeic Acid Due to Ca2＋ MobilizationInhibition Via cAMP-Dependent Inositol-1, 4, 5-Trisphosphate Receptor Phosphorylation

In this study, we investigated the effects of caffeic acid (CAFA), a phenolic acid, on Ca(2＋)-antagonistic cyclic nucleotides associated with the phosphorylation of inositol 1,4,5-trisphosphate receptor (IP3R) and vasodilator-stimulated phosphoprotein (VASP) and the thromboxane A2 (TXA2)-associated microsomal cyclooxygenase-1 (COX-1) activity in collagen (10 μg/mL)-stimulated platelet aggregation. Washed platelets (10(8)/mL) obtained from Sprague-Dawley rats (6-7 weeks old, male) were preincubated for 3 minutes at 37℃ in the presence of 2 mM exogenous CaCl2 with or without CAFA or other materials, stimulated with collagen (10 μg/mL) for 5 minutes, then used to determine the levels of intracellular cytosolic Ca(2＋) ([Ca(2＋)]i), TXA2, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), COX-1 activity, VASP and IP3R phosphorylation. CAFA dose-dependently inhibited collagen-induced platelet aggregation and suppressed the production of TXA2, an aggregation-inducing autacoid associated with the strong inhibition of COX-1 in platelet microsomes exhibiting cytochrome C reductase activity. CAFA dose-dependently inhibited collagen-elevated [Ca(2＋)]i mobilization, which was increased by a cAMP-dependent protein kinase (A-kinase) inhibitor, Rp-8-Br-cAMPS, but not a cGMP-dependent protein kinase (G-kinase) inhibitor, Rp-8-Br-cGMPS. In addition, CAFA significantly increased the formation of cAMP and cGMP, intracellular Ca(2＋)-antagonists that function as aggregation-inhibiting molecules. CAFA increased IP3R (320 kDa) phosphorylation, indicating the inhibition of IP3-mediated Ca(2＋) release from internal stores (i.e. the dense tubular system) via the IP3R on collagen-activated platelets. Furthermore, CAFA-induced IP3R phosphorylation was strongly inhibited by an A-kinase inhibitor, Rp-8-Br-cAMPS, but only mildly inhibited by a G-kinase inhibitor, Rp-8-Br-cGMPS. These results suggest that the inhibition of [Ca(2＋)]i mobilization by CAFA is resulted from the cAMP/A-kinase-dependent phosphorylation of IP3R. CAFA elevated the phosphorylation of VASP-Ser(157), an A-kinase substrate, but not the phosphorylation of VASP-Ser(239), a G-kinase substrate. We demonstrate that CAFA increases cAMP and subsequently phosphorylates both IP3R and VASP-Ser(157) through A-kinase activation to inhibit [Ca(2＋)]i mobilization and TXA2 production via the inhibition of the COX-1 activity. These results strongly indicate that CAFA is a potent beneficial compound that elevates the level of cAMP-dependent protein phosphorylation in collagen-platelet interactions, which may result in the prevention of platelet aggregation-mediated thrombotic diseases.

Hydrogen sulfide in regulation of frog myocardium contractility

Hydrogen sulfide (H2S) is an endogenously synthesized gaseous molecule which, along with nitric oxide and carbon monoxide, induces a number of effects in cardiovascular system under normal and pathological conditions. In the present work, the effects and underlying mechanisms of the H2S donor sodium hydrosulfide (NaHS) on the isometric force of frog myocardium contraction have been studied. NaHS at the concentration of 100 μM induced negative inotropic effect and reduced the maximum velocity of the contraction and relaxation of the isolated ventricle strips. The substrate of H2S synthesis, L-cysteine (200 μM and 1 mM), induced the same effect, while the inhibitors of cystathionin-γ-lyase, the H2S-producing enzyme in heart, β-cyanoalanine (500 μM) and propargylglycine (500 μM), increased the amplitude of contraction. Inhibition of cystathionin-γ-lyase by β-cyanoalanine prevented the negative inotropic effect of L-cysteine. After the inhibition of adenylate cyclase by MDL-12,330A (3 μM) or phosphodiesterases by IBMX (200 μM), the effect of NaHS was less than that in the control. In the presence of membrane-penetrating analogous of cAMP, 8Br-cAMP (100 μM) and pCPT-cAMP (100 μM), the negative inotropic effect of NaHS was completely retained. The effect of NaHS significantly decreased after preliminary application of the NO donor, SNAP (10 μM), and did not change after the inhibition of NO synthases by L-NAME (100 μM). The results suggest the possibility of endogenous synthesis of H2S in frog myocardium and regulation of its contractility by the activation of phosphodiesterases hydrolyzing cAMP, which leads to a decrease in the activation of cAMP-dependent protein kinases and phosphorylation of voltage-dependent L-type Ca channels. As a result, the reduction of calcium entry into cardiomyocytes decreases the contractility of frog myocardium.

CAMP-dependent protein kinase regulates post-translational processing and expression of complex I subunits in mammalian cells

Work is presented on the role of cAMP-dependent protein phosphorylation in post-translational processing and biosynthesis of complex I subunits in mammalian cell cultures. PKA-mediated phosphorylation of the NDUFS4 subunit of complex I promotes in cell cultures in vivo import/maturation in mitochondria of the precursor of this protein. The import promotion appears to be associated with the observed cAMP-dependent stimulation of the catalytic activity of complex I. These effects of PKA are counteracted by activation of protein phosphatase(s). PKA and the transcription factor CREB play a critical role in the biosynthesis of complex I subunits. CREB phosphorylation, by PKA and/or CaMKs, activates at nuclear and mitochondrial level a transcriptional regulatory cascade which promotes the concerted expression of nuclear and mitochondrial encoded subunits of complex I and other respiratory chain proteins.

Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399-4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a K(a) of (2.44 +/- 0.09) x 10(5) m(-1) with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a K(a) of (5.83 +/- 0.05) x 10(4) m(-1) with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca(2+) sensitivity in that in the presence of Ca(2+) complex formation was reduced.

Removal of the N-terminal Extension of Cardiac Troponin I as a Functional Compensation for Impaired Myocardial β-Adrenergic Signaling

Although beta-adrenergic stimuli are essential for myocardial contractility, beta-blockers have a proven beneficial effect on the treatment of heart failure, but the mechanism is not fully understood. The stimulatory G protein alpha-subunit (Gsalpha) couples the beta-adrenoreceptor to adenylyl cyclase and the intracellular cAMP response. In a mouse model of conditional Gsalpha deficiency in the cardiac muscle (Gsalpha-DF), we demonstrated heart failure phenotypes accompanied by increases in the level of a truncated cardiac troponin I (cTnI-ND) from restricted removal of the cTnI-specific N-terminal extension. To investigate the functional significance of the increase of cTnI-ND in Gsalpha-DF cardiac muscle, we generated double transgenic mice to overexpress cTnI-ND in Gsalpha-DF hearts. The overexpression of cTnI-ND in Gsalpha-DF failing hearts increased relaxation velocity and left ventricular end diastolic volume to produce higher left ventricle maximum pressure and stroke volume. Supporting the hypothesis that up-regulation of cTnI-ND is a compensatory rather than a destructive myocardial response to impaired beta-adrenergic signaling, the aberrant expression of beta-myosin heavy chain in adult Gsalpha-DF but not control mouse hearts was reversed by cTnI overexpression. These data indicate that the up-regulation of cTnI-ND may partially compensate for the cardiac inefficiency in impaired beta-adrenergic signaling.

Molecular Mechanisms Determining Sperm Motility Initiation in Two Sparids (Sparus aurata and Lithognathus mormyrus)

Molecular mechanisms involved in sperm motility initiation in two sparids (Sparus aurata and Lithognathus mormyrus) have been studied. Our comparative study demonstrates that osmolality is the key signal in sperm motility activation in both species, whereas K(+) and Ca(2+) do not have any role. The straight-line velocity that resulted, however, was significantly different when measured in sperm activated with non-ionic and/or calcium-free solutions with respect to that measured in seawater-activated sperm. In both species, motility initiation depends on cAMP-dependent protein phosphorylation. The phosphorylation/dephosphorylation patterns that resulted in gilthead and striped sea bream were quite different. In gilthead sea bream, the phosphorylated proteins have molecular weights of 174, 147, 138, 70, and 9-15 kDa, whereas the dephosphorylated proteins have molecular weights of 76, 57, and 33 kDa. In striped sea bream, phosphorylation after sperm motility activation occurred on proteins of 174, 147, 103, 96, 61, 57, and 28 kDa, whereas only one protein of 70 kDa resulted from dephosphorylation. Matrix-assisted laser desorption ionization-time of flight analyses allowed identification of the following proteins: In gilthead sea bream, the 9-15 kDa proteins that were phosphorylated after motility activation include an A-kinase anchor protein (AKAP), an acetyl-coenzyme A synthetase, and a protein phosphatase inhibitor, and in striped sea bream, 103- and 61-kDa proteins that were phosphorylated after motility activation were identified as a phosphatase (myotubularin-related protein 1) and a kinase (DYRK3), respectively.

Region‐specific tolerance to cocaine‐regulated cAMP‐dependent protein phosphorylation following chronic self‐administration

Chronic cocaine self-administration can produce either tolerance or sensitization to certain cocaine-regulated behaviours, but whether differential alterations develop in the biochemical response to cocaine is less clear. We measured cocaine-induced phosphorylation of multiple cAMP-dependent and -independent protein substrates in mesolimbic dopamine terminal regions following chronic self-administration. Changes in self-administering rats were compared to changes produced by passive yoked injection to identify reinforcement-related regulation, whereas acute and chronic yoked groups were compared to identify the development tolerance or sensitization in the biochemical response to cocaine. Microwave-fixed brain tissue was collected immediately following 4 h of intravenous cocaine administration, and subjected to Western blot analysis of phosphorylated and total protein substrates. Chronic cocaine produced region- and substrate-specific tolerance to cAMP-dependent protein phosphorylation, including GluR1(S845) phosphorylation in striatal and amygdala subregions and NR1(S897) phosphorylation in the CA1 subregion of the hippocampus. Tolerance also developed to cAMP-independent GluR1(S831) phosphorylation in the prefrontal cortex. In contrast, sensitization to presynaptic regulation of synapsin(S9) phosphorylation developed in the hippocampal CA3 subregion while cAMP-dependent tyrosine hydroxylase(S40) phosphorylation decreased in striatal dopamine terminals. Cocaine-induced ERK and CREB(S133) phosphorylation were dissociated in many brain regions and failed to develop either tolerance or sensitization with chronic administration. Positive reinforcement-related correlations between cocaine intake and protein phosphorylation were found only in self-administering animals, while negative dose-related correlations were found primarily with yoked administration. These regional- and substrate-specific adaptations in cocaine-induced protein phosphorylation are discussed in view of their potential impact on the development of cocaine addiction.

Increase in intracellular pH induces phosphorylation of axonemal proteins for activation of flagellar motility in starfish sperm

Increased intracellular pH ([pH]i) activates dynein in sea urchin and mammalian sperm and induces activation of flagellar motility. It is thought that cAMP-dependent protein phosphorylation is associated with motility activation through increasing [pH]i, but little attention has been given to the cAMP-independent phosphorylation also induced by the [pH]i increase. The present study demonstrates that the increase in [pH]i in starfish sperm induces the phosphorylation of axonemal proteins and activation of flagellar motility independently of cAMP. Flagellar motility of intact sperm was activated when the [pH]i was raised by addition of NH4Cl. Histidine, which is known to activate motility of starfish sperm, also raised the [pH]i during the motility activation. In addition, motility of demembranated sperm flagella was activated in a pH-dependent manner without cAMP. These results indicate that in starfish sperm it is the increase in [pH]i that induces activation of flagellar motility. Moreover, phosphorylation of axonemal proteins (of molecular mass 25, 32 and 45 kDa) was observed during the pH-dependent and cAMP-independent motility activation of demembranated sperm. This suggests that the increase in [pH]i regulates flagellar motility via cAMP-independent phosphorylation of axonemal proteins.

In Vitro Phosphorylation by cAMP-dependent Protein Kinase Up-regulates Recombinant Saccharomyces cerevisiae Mannosylphosphodolichol Synthase

DPM1 is the structural gene for mannosylphosphodolichol synthase (i.e. Dol-P-Man synthase, DPMS) in Saccharomyces cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that 31-kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS141) that can be phosphorylated by cAMP-dependent protein kinase (PKA). We have been studying the up-regulation of DPMS activity by protein kinase A-mediated phosphorylation in higher eukaryotes, and used the recombinant DPMS from S. cerevisiae in this study to advance our knowledge further. DPMS catalytic activity was indeed enhanced severalfold when the recombinant protein was phosphorylated in vitro. The rate as well as the magnitude of catalysis was higher with the phosphorylated enzyme. A similar increase in the catalytic activity was also observed when the in vitro phosphorylated recombinant DPMS was assayed as a function of increasing concentrations of exogenous dolichylmonophosphate (Dol-P). Kinetic studies indicated that there was no change in the Km for GDP-mannose between the in vitro phosphorylated and control recombinant DPMS, but the Vmax was increased by 6-fold with the phosphorylated enzyme. In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (kcat) and enzyme efficiency (kcat/Km). SDS-PAGE followed by autoradiography of the 32P-labeled DPMS detected a 31-kDa phosphoprotein, and immunoblotting with anti-phosphoserine antibody established the presence of a phosphoserine residue in in vitro phosphorylated recombinant DPMS. To confirm the phosphorylation activation of recombinant DPMS, serine 141 in the consensus sequence was replaced with alanine by PCR site-directed mutagenesis. The S141A DPMS mutant exhibited more than half-a-fold reduction in catalytic activity compared with the wild type when both were analyzed after in vitro phosphorylation. Thus, confirming that S. cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation signal, and the phosphorylation target is serine 141.

Augmented Ciliary Reorientation Response and cAMP-dependent Protein Phosphorylation Induced by Glycerol in Triton-extracted Paramecium

In the presence of 30% glycerol, the cilia of a permeabilized cell model from Paramecium exhibit dynamic orientation changes while displaying only a restricted cyclic beating with a very small amplitude. The direction of cilia under these conditions corresponds to the direction of the effective power stroke of cilia beating in the absence of glycerol, i.e., pointing posteriorly in the absence of Ca2+ and anteriorly at > 10(-6) M Ca2+. Ciliary reorientation toward the posterior in response to the removal of Ca2+ is particularly conspicuous; all the cilia become predominantly pointing to the posterior end all through their beating phases. Previous studies suggested that the effect of glycerol is caused through modification of cAMP-dependent protein phosphorylation. To determine whether glycerol in fact affects ciliary reorientation through changes in protein phosphorylation, here we examined protein phosphorylation in the axonemes. Glycerol stimulated cAMP-induced phosphorylation of 29-kDa and 65-kDa proteins. The stimulation of phosphorylation was found to be partly due to the inhibition of endogenous phosphodiesterase (PDE), and partly due to the inhibition of the dephosphorylation of the 29-kDa and 65-kDa phosphoproteins within the axoneme. Thus glycerol appears to cause predominant posterior orientation of cilia by stimulating cAMP-dependent phosphorylation on those proteins. In addition, glycerol appears to inhibit ciliary beating through inhibition of dynein ATPase.

Dephosphorylation of Tctex2-related dynein light chain by type 2A protein phosphatase

Sperm flagellar movements are regulated by cAMP-dependent protein phosphorylation. Tctex2-related light chain of outer arm dynein is a well-defined phosphorylated protein that is phosphorylated at activation of sperm motility. Here, the protein phosphatase that dephosphorylates Tctex2-related dynein light chain (LC2) has been characterized in salmonid fish sperm. Most of the phosphatase activity against LC2 is found in Triton-soluble fraction of flagella but trace extent of the activity is retained in the axoneme. The dephosphorylation of LC2 is inhibited by okadaic acid at more than 1 nM, whereas that of dynein α heavy chain is inhibited at more than 10 nM. The addition of Ca 2+ gives no direct effect on LC2 dephosphorylation, but it accelerates the dephosphorylation of the regulatory subunit of cAMP-dependent protein kinase, resulting in the decrease of LC2 phosphorylation. The activity to dephosphorylate the LC2 is separated by MonoQ ion-exchange column chromatography along with the immunoreactivity to the antibody against the catalytic subunit of type 2A protein phosphatase. These results suggest that LC2 is dephosphorylated by type 2A protein phosphatase and that dynein α heavy chain and the regulatory subunit of cAMP-dependent protein kinase are dephosphorylated by other types of protein phosphatases.

Cyclic AMP-dependent protein kinase is involved in morphogenesis of Aspergillus niger.

The cAMP signal transduction pathway controls many processes in fungi. The pkaR gene, encoding the regulatory subunit (PKA-R) of cAMP-dependent protein kinase (PKA), was cloned from the industrially important filamentous fungus Aspergillus niger. To investigate the involvement of PKA in morphology of A. niger, a set of transformants which overexpressed pkaR or pkaC (encoding the catalytic subunit of PKA) either individually or simultaneously was prepared as well as mutants in which pkaR and/or pkaC were disrupted. Strains overexpressing pkaR or both pkaC and pkaR could not be distinguished from the wild-type, suggesting that regulation of PKA activity is normal in these strains. Absence of PKA activity resulted in a two- to threefold reduction in colony diameter on plates. The most severe phenotype was observed in the absence of PKA-R, i.e., very small colonies on plates, absence of sporulation and complete loss of growth polarity during submerged growth. Suppressor mutations easily developed in the DeltapkaR mutant and one of these mutants appeared to lack PKA-C activity. These data suggest that cAMP-dependent protein phosphorylation in A. niger regulates growth polarity and formation of conidiospores.

Capacitation induces cyclic adenosine 3',5'-monophosphate-dependent, but apoptosis-unrelated, exposure of aminophospholipids at the apical head plasma membrane of boar sperm cells.

The capacitating agent bicarbonate/CO(2) has been shown to induce profound changes in the architecture and dynamics within the sperm's plasma membrane lipid bilayer via a cAMP-dependent protein phosphorylation signaling pathway. Here we have investigated the effect of bicarbonate on surface exposure of endogenous aminophospholipids in boar spermatozoa, detecting phosphatidylserine (PS) with fluorescein-conjugated annexin V and phosphatidylethanolamine (PE) with fluorescein-conjugated streptavidin/biotinylated Ro-09-0198. Flow cytometric analyses revealed that incubation with 15 mM bicarbonate induced 30%-70% of live acrosome-intact cells to expose PE very rapidly; this exposure was closely related to a decrease in lipid packing order as detected by enhanced binding of merocyanine 540. PS exposure was detectable in the same proportion of cells, though its expression was slower. Confocal microscopy revealed that exposure of aminophospholipids in intact cells was restricted to the anterior acrosomal region of the head plasma membrane. Aminophospholipid exposure, merocyanine stainability, and a subsequent migration of cholesterol to the apical region of the head plasma membrane, were all under the control of the cAMP-dependent protein phosphorylation pathway. The close coupling of decreased lipid packing order with exposure of PE led us to conclude that bicarbonate was inducing phospholipid scrambling (i.e., collapse of asymmetric transverse distribution), and that the scrambling was a prerequisite for cholesterol relocation. There was no evidence whatever that the bicarbonate-induced scrambling was an apoptotic process. It was not accompanied by major loss of viability or by DNA degeneration or by loss of mitochondrial function, and it could not be blocked by the broad-specificity caspase inhibitors zVAD-fmk and BocD-fmk. In the absence of bicarbonate, scrambling could not be induced by the apoptotic agents UV, staurosporine, or cycloheximide. Bicarbonate-induced phospholipid scrambling thus appears to be an important and early physiological event in the capacitation process.

Tyrosine Phosphorylation and Dephosphorylation Associated with Motility of Hamster Spermatozoa

Although sperm motility is regulated through cAMP-dependent protein phosphorylation cascade, the mechanism remains obscure. Recently it has been suggested that cAMP-dependent tyrosine phosphorylation plays an important role in the regulatory mechanisms of sperm motility. In the present experiments, fourteen flagellar proteins that ranged from 10k-Da to 120k-Da were detected as tyrosine phosphoprotein associated with the motility of hamster spermatozoa. 100k-Da, 75k-Da, 70k-Da, 60k-Da, 45k-Da, 30k-Da and 10k-Da proteins were phosphorylated when sperm motility was activated. Among them, 30k-Da and 10k-Da proteins were phosphorylated in a cAMP-dependent manner. On the other hand, 20k-Da protein was dephosphorylated when spermatozoa were activated, but it was phosphorylated in immotile spermatozoa. When activated spermatozoa were hyperactivated, 120k-Da, 115k-Da, 80k-Da, 50k-Da 40k-Da and 16k-Da proteins were phosphorylated. Form these results, it was assumed that activation of hamster sperm motility was regulated through tyrosine phosphorylation and dephosphorylation in addition to serine phosphorylation.