Creatine kinase regulation by reversible phosphorylation in frog muscle

Creatine kinase (CK) was analyzed from skeletal muscle of wood frogs, Rana sylvatica, a species that survives natural whole body freezing during the winter months. Muscle CK activity increased by 35% and apparent K_m creatine decreased by 29% when frogs froze. Immunoblotting analysis showed that this activity increase was not due to a change in total CK protein. Frog muscle CK was regulated by reversible protein phosphorylation; in vitro incubations with ³²P-ATP under conditions that facilitated the actions of various protein kinases (PKA, PKG, PKC, CaMK or AMPK) resulted in immunoprecipitation of ³²P-labeled CK. Furthermore, incubations that stimulated CaMK or AMPK altered CK kinetics. Incubation under conditions that facilitated protein phosphatases (PP2B or PP2C) reversed these effects. Phosphorylation of CK increased activity, whereas dephosphorylation decreased activity. Ion-exchange chromatography revealed that two forms of CK with different phosphorylation states were present in muscle; low versus high phosphate forms dominated in muscle of control versus frozen frogs, respectively. However, CK from control versus frozen frogs showed no differences in susceptibility to urea denaturation or sensitivity to limited proteolysis by thermolysin. The increased activity, increased substrate affinity and altered phosphorylation state of CK in skeletal muscle from frozen frogs argues for altered regulation of CK under energy stress in ischemic frozen muscle.     

Oocyte maturation is inhibited by adenosine in the presence of follicle-stimulating hormone

Considerable evidence implicates cyclic 3', 5' adenosine monophosphate (AMP) in the maintenance of meiotic arrest of mammalian oocytes. Since this laboratory previously found that adenosine augmented follicle-stimulating hormone (FSH)-stimulated accumulation of cyclic AMP in oocyte-cumulus-complexes (OCC), in the present studies we investigated the possibility that adenosine inhibits maturation of oocytes. In rat OCC cultured in the presence of FSH, adenosine markedly inhibited oocyte maturation in a dose-dependent and biphasic manner. Maximum inhibition of oocyte maturation was seen with 1-30 microM adenosine in the presence of FSH, and half-maximal inhibition occurred with less than 0.3 microM adenosine. High levels of adenosine (100 microM) did not inhibit oocyte maturation in the presence of FSH. In the absence of FSH, adenosine showed little effect on oocyte maturation in the present studies, but increased the maximum inhibition of oocyte maturation produced by FSH approximately twofold. Like adenosine, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine 5'-monophosphate (AMP) also inhibited oocyte maturation; whereas adenine, guanosine, inosine, and hypoxanthine were inactive at equivalent levels. The metabolism-resistant adenosine analog (2-chloroadenosine) was as active an inhibitor as adenosine. Inhibition produced by the adenine nucleotides may have been direct or due to conversion to adenosine by extracellular nucleotidases. The concentration dependence and purine specificity for inhibition of oocyte maturation are characteristic of an adenosine receptor-mediated process, but direct evidence for such a mechanism was not shown. The effective concentration of adenosine for inhibition of oocyte maturation is within the range of reported levels of adenosine in biological tissues and fluids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction between photoexcited rhodopsin and peripheral enzymes in frog retinal rods. Influence on the postmetarhodopsin II decay and phosphorylation rate of rhodopsin

The major peripheral and soluble proteins in frog rod outer segment preparations, and their interactions with photoexcited rhodopsin, have been compared to those in cattle rod outer segments and found to be similar in both systems. In particular the GTP-binding protein (G) has the same subunit composition, the same abundance relative to rhodopsin (1/10) and it undergoes the same light and nucleotide-dependent interactions with rhodopsin in both preparations. Previous work on cattle rod outer segments has shown that photoexcited rhodopsin (R*), in a state identified with metarhodopsin II, associates with the G protein as a first step to the light-activated GDP/GTP exchange on G. The complex R*-G is stable in absence of GTP, but is rapidly dissociated by GTP owing to the GDP/GTP exchange reaction. Low bleaching extents (less than 10% R*) in absence of GTP therefore create predominantly R*-G complexes, whereas bleaching in presence of GTP creates free R*. We report here that, under conditions of complexed R*, two reactions of R* in frog rod outer segments are highly perturbed as compared to free R*: (a) the spectral decay of metarhodopsin II (MII) into later photoproducts, and (b) the phosphorylation of R* by an ATP-dependent protein kinase. a) The spectral measurements have been performed using linear dichroism on oriented frog rod outer segments; this technique allows discrimination between MII and later photoproducts absorbing at the same wavelength. Association of R* with G leads to a strong reduction of the amount of MIII formed and to an acceleration of the decay of MIII. Furthermore, MII is significantly stabilized, in agreement with the hypothesis that MII is the intermediate which binds to G. b) The phosphorylation of R* is strongly inhibited under conditions of R*-G complex formation as compared to free R*. Interferences between reactions at the three sites involved in R* are discussed: the retinal binding site in the hydrophobic core is sensitive to the presence of GTP-binding protein at its binding site on the cytoplasmic surface of R*; the kinase and the GTP-binding protein compete for access to their respective binding sites, both located on the surface of R*. We also observed a slow and nucleotide-dependent light-induced binding of a protein of molecular weight 50 000, which we consider as the equivalent of the 48 000 Mr light-dependent protein previously identified in cattle rod outer segments.     

Transient state in the phosphorylation of sodium- and potassium- transport adenosine triphosphatase by adenosine triphosphate

The rate of phosphorylation of sodium and potassium ion-transport adenosine triphosphatase by 10 microM [gamma-32P]ATP was much slower with Ca2+ than with Mg2+ (0.13-10 mM) in the presence of 16 to 960 mM Na+ at 0 degrees C and pH 7.4. In the presence of a fixed concentration of Mg2+ or Ca2+, the rate became slower with increasing Na+ concentration. When the Na+ concentration was fixed, the rate became slower with decreasing divalent cation concentration. Sodium ions appear to antagonize the divalent cation in the phosphorylation to slow its rate. In the presence of 1 mM Ca2+ and 126 or 270 mM Na+, the rate was slow enough to permit the manual addition of a chasing solution at various times before the phosphorylation reached the steady state. Therefore, we studied the time-dependent change of the sensitivity to ADP or to K+ of the phosphoenzyme by a chase with unlabeled ATP containing ADP or K+ during the time range from the transient to the steady state of the phosphorylation. The ADP sensitivity decreased and the K+ sensitivity increased with the progress of the phosphorylation. With 270 mM Na+, the phosphoenzyme found at 1 s, when its amount was 5.5% of the maximum level, was virtually completely sensitive to ADP. Under these conditions, it was concluded that the form of the phosphoenzyme initially produced from the enzyme.ATP complex has ADP sensitivity and that the phosphoenzyme acquires K+ sensitivity later. The initially produced ADP-sensitive phosphoenzyme partially lost its normal instability and sensitivity upon adding a chelating agent, probably because of dissociation of a divalent cation from the phosphoenzyme.     

Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning

Loss of tuberin, the product of TSC2 gene, increases mammalian target of rapamycin (mTOR) signaling, promoting cell growth and tumor development. However, in cells expressing tuberin, it is not known how repression of mTOR signaling is relieved to activate this pathway in response to growth factors and how hamartin participates in this process. We show that hamartin colocalizes with hypophosphorylated tuberin at the membrane, where tuberin exerts its GTPase-activating protein (GAP) activity to repress Rheb signaling. In response to growth signals, tuberin is phosphorylated by AKT and translocates to the cytosol, relieving Rheb repression. Phosphorylation of tuberin at serines 939 and 981 does not alter its intrinsic GAP activity toward Rheb but partitions tuberin to the cytosol, where it is bound by 14-3-3 proteins. Thus, tuberin bound by 14-3-3 in response to AKT phosphorylation is sequestered away from its membrane-bound activation partner (hamartin) and its target GTPase (Rheb) to relieve the growth inhibitory effects of this tumor suppressor.     

Regulation of hexokinase by reversible phosphorylation in skeletal muscle of a freeze-tolerant frog

Hexokinase (HK) was isolated from hind leg skeletal muscle of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K(m) and V(max)) of HK showed significant increases in K(m) glucose (from 144 ± 4.4 to 248 ± 1 2.0 μM) and K(m) ATP (from 248 ± 8.5 to 330 ± 20.9 μM), as well as a decrease in V(max) (from 86.1 ± 0.40 to 52 ± 0.49 mUmg(-1) of protein) in frogs following freezing exposure, indicating lower affinity for HK substrates and lower enzyme activity in this state. Subsequent analyses indicated that differential phosphorylation of HK between the two states was responsible for the altered kinetic properties. HK was analyzed by SDS-PAGE; phosphoprotein staining revealed a 33% decrease in phosphate content of HK from frozen frogs but immunoblotting showed no change in total HK protein content. Muscle extracts from control and frozen frogs were incubated with ions and second messengers to stimulate the actions of protein kinases and protein phosphatases, with results indicating that HK can be phosphorylated by protein kinases A and C, and AMP-activated protein kinase, and can be dephosphorylated by protein phosphatases 1, 2A and 2C. The data indicate that in control frogs, HK is in a higher phosphate form and displays a high substrate affinity and high activity, whereas in frozen frogs HK is less phosphorylated, with lower substrate affinity and lower activity. Studies also showed that HK affinity for ATP decreases further in response to low temperature, but that high cryoprotective glucose concentrations can prevent these changes in affinity. Finally, the activity and structure of HK from frozen frogs is more sensitive to non-compatible osmolytes than the enzyme in control frogs.     

Relaxation of smooth muscle by cardiodilatin/atrial natriuretic peptide is inhibited by cAMP-dependent phosphorylation

Cardiodilatins/atrial natriuretic peptides (CDD/ANP) exhibit a common amino acid sequence: Arg101-Arg102-Ser103-Ser104. Cyclic AMP-dependent phosphorylation of Ser104 of atrial peptides with [gamma-32P]ATP enables rapid identification of cardiac hormones. The biological activity of in vitro phosphorylated cardiodilatin (CDD-28/alpha-hANP) is dramatically altered compared to the unphosphorylated peptide: the vaso-relaxant effect of cardiodilatin 28 is inhibited upon phosphorylation.     

Control of light-activated phosphorylation in frog photoreceptor membranes.

In this paper, we examine some factors which regulate the efficiency of light in activating rhodopsin phosphorylation. We have measured phosphate incorporation after illumination in suspensions of bullfrog rod outer segments incubated with [gamma-32P]ATP. We observed that delaying ATP addition after illumination causes maximum phosphate incorporation to decrease 80% within 2 h. This decay occurs in urea-treated, extracted rod outer segment membranes. The decay of the light effect is not influenced by regeneration of opsin to rhodopsin or the presence of long-lived photoproducts. However, regeneration of opsin increases the amount of phosphorylation initiated by a second exposure to light. Further phosphorylation can also occur after phosphate groups have been removed from the membranes by dephosphorylation. Finally, we have confirmed our earlier observation that small amounts of light (bleaching less than 5% of the rhodopsin present) are more effective, by tenfold, in initiating phosphorylation than are larger amounts.