A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.     

Protein kinases in human renal cell carcinoma and renal cortex. A comparison of isozyme distribution and of responsiveness to adenosine 3':5'-cyclic monophosphate.

Two major isozyme forms of cyclic AMP-dependent protein kinase (termed protein kinase I and II according to their order of elution from DEAE-cellulose) were resolved by DEAE-cellulose chromatography of extracts from human renal cortex and renal cell carcinoma. The ratio between protein kinase I and protein kinase II in carcinoma extracts was about twice that in extracts of renal cortex. The total soluble cyclic AMP-dependent protein kinase activity was similar in extracts from the normal and malignant tissue. Protein kinase isozymes prepared from renal cortex or carcinoma were highly dependent on cyclic AMP for activity under appropriate assay conditions, were activated to the same degree by various concentrations of cyclic AMP, and had similar affinity for the nucleotide, indicating that the mechanism for regulation of protein kinase activity by cyclic AMP was intact for the tumor kinases. The kinetics of endogenous phosphorylation of protein kinase II was similar for enzyme derived from normal or malignant tissue.     

Regulation of multiple forms of cyclic nucleotide phosphodiesterase from bovine hypothalamus: New factors modulating enzyme activity

Studies of bovine hypothalamic cyclic nucleotide phosphodiesterase (PDE) indicate the presence of several peaks of PDE activity, distinguishable by DEAE-cellulose column chromatography, displaying different substrate specificities, kinetic behavior, and regulatory properties. Evidence is presented that chromatographically separated forms of PDE activity are subject to control by Ca²⁺-calmodulin, cyclic nucleotides, limited proteolysis, reagents affecting sulfhydryl groups, and neurohormone “C”—one of several new cardioactive compounds isolated from hypothalamic magnocellular nuclei of animals—in a complex substrate-specific and concentration-dependent manner. Of particular interest is the finding that each of the forms of cGMP PDE, being Ca²⁺/calmodulin-dependent, possesses sensitivity to activation by cAMP, especially under conditions favoring the oxidation of thiol groups of PDE, resulting in a loss in responsiveness of the enzyme to the activation by calmodulin. This effect appears to be relatively stable but readily reversible by sulfhydryl reducing reagents, which restore both the cGMP PDE sensitivity to competitive inhibition by cAMP and the responsiveness of the enzyme to activation by calmodulin. A reinterpretation of the regulatory properties of multiple forms of PDE is proposed. Special Issue dedicated to Dr. Eugene Kreps.     

Activation of mammalian cyclic AMP phosphodiesterases by trypsin.

BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) as analyzed by linear sucrose gradient fractionation; a 3.6-S form (peak I) and a 6.7-S form (peak II). Peak I is specific for cyclic AMP as substrate and displays Michaelis-Menten kinetics with an apparent Km of 2--3 micrometer. Peak II hydrolyzes cyclic GMP and displays anomalous kinetics for cyclic AMP hydrolysis. The activity of isolated peak II for cyclic AMP is increased by storage at 4 degrees C, treatment with trypsin, or treatment with rat brain and BHK fibroblast activator proteins. The activity of isolated peak I is unaffected by these conditions. Linear sucrose gradient fractionation demonstrates that activation of peak II by trypsin leads to the formation of a 3.6-S cyclic AMP-specific enzyme form, possibly peak I. In contrast to BHK fibroblasts (and most other mammalian tissues), rat uterus contains only one form of cyclic nucleotide phosphodiesterase on linear sucrose gradients, a 7-S form capable of hydrolyzing both cyclic AMP and cyclic GMP. Treatment of rat uterine supernatant with trypsin leads to the appearance of a 4-S, cyclic AMP-specific form with properties similar to that of BHK peak I. These data suggest that the kinetically complex, higher molecular weight cyclic nucleotide phosphodiesterases may consist of more than one catalytically active site and that multiple forms of the enzyme arise through dissociative mechanisms, possibly as a means of in vivo regulation.     

Activation of the cAMP-specific phosphodiesterase PDE4D3 by phosphorylation. Identification and function of an inhibitory domain.

Splicing variants of type 4 phosphodiesterases (PDE4) are regulated by phosphorylation. In these proteins, a conserved region is located between the amino-terminal domain, which is the target for phosphorylation, and the catalytic domain. Previous studies have indicated that nested deletions encompassing this region cause an increase in catalytic activity, suggesting this domain exerts an inhibitory constraint on catalysis. Here, we have further investigated the presence and function of this domain. A time-dependent increase in hydrolytic activity was observed when PDE4D3 from FRTL-5 cells was incubated with the endoproteinase Lys-C. The activation was abolished by protease inhibitors and was absent when a phosphorylated enzyme was used. Western blot analysis with PDE4D-specific antibodies indicated the Lys-C treatment separates the catalytic domain of PDE4D3 from the inhibitory domain. Incubation with antibodies recognizing an epitope within this domain caused a 3- to 4-fold increase in activity of native or recombinant PDE4D3. Again, PDE activation by these antibodies had properties similar to, and not additive with, the activation by protein kinase A phosphorylation. An interaction between the inhibitory domain and both regulatory and catalytic domains of PDE4D3 was detected by the yeast two-hybrid system. Mutations of Ser54 to Ala in the regulatory domain decreased or abolished this interaction, whereas mutations of Ser54 to the negatively charged Asp strengthened it. These data strongly support the hypothesis that an inhibitory domain is present in PDE4D and that phosphorylation of the regulatory domain causes activation of the enzyme by modulating the interaction between inhibitory and catalytic domains.     

Structural differences between repressed and derepressed forms of p60c-src.

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.     

The separation of the heme and apoheme forms of soluble guanylate cyclase

Two forms of soluble quanylate cyclase from mammalian tissues can be separated on DEAE Sephacel or Blue Sepharose CL-6B. The two forms, referred to as peak I or peak II, migrate identically during electrophoresis on polyacrylamide gels in the presence or absence of Na-dodecyl-SO₄. Peak I is markedly stimulated by sodium nitroprusside and is the heme-containing form of guanylate cyclase. Peak II is only weakly stimulated by nitroprusside and contains no heme absorbance. In fresh tissue extracts, peak I is the predominant form, but it can be converted to peak II by treatments (pH 5.0, storage at 4°C) that result in the loss of the heme absorbance from the enzyme. Peak II is not formed from peak I by proteolysis.     

Proteolytic regulation of the mitochondrial cAMP-dependent protein kinase

The mitochondrial cAMP-dependent protein kinase (PKA) is activatable in a cAMP-independent fashion. The regulatory (R) subunits of the PKA holoenzyme (R(2)C(2)), but not the catalytic (C) subunits, suffer proteolysis upon exposure of bovine heart mitochondria to digitonin, Ca(2+), and a myriad of electron transport inhibitors. Selective loss of both the RI- and RII-type subunits was demonstrated via Western blot analysis, and activation of the C subunit was revealed by phosphorylation of a validated PKA peptide substrate. Selective proteolysis transpires in a calpain-dependent fashion as demonstrated by exposure of the R and C subunits of PKA to calpain and by attenuation of R and C subunit proteolysis in the presence of calpain inhibitor I. By contrast, exposure of mitochondria to cAMP fails to promote R subunit degradation, although it does result in enhanced C subunit catalytic activity. Treatment of mitochondria with electron transport chain inhibitors rotenone, antimycin A, sodium azide, and oligomycin, as well as an uncoupler of oxidative phosphorylation, also elicits enhanced C subunit activity. These results are consistent with the notion that signals, originating from cAMP-independent sources, elicit enhanced mitochondrial PKA activity.     

Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells.

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.     

Autophosphorylation of the type II calmodulin-dependent protein kinase is essential for formation of a proteolytic fragment with catalytic activity. Implications for long-term synaptic potentiation

Autophosphorylation plays an essential role in proteolytic activation of the type II calmodulin-dependent protein kinase (CaM kinase II). Limited proteolysis of CaM kinase II by trypsin, alpha-chymotrypsin, and Ca2+-stimulated neutral protease (calpain) yielded a catalytically active kinase fragment only when the holoenzyme was autophosphorylated prior to proteolysis. Slightly larger, inactive fragments were obtained from nonphosphorylated CaM kinase II, regardless of whether Ca2+/calmodulin or Mg2+/ATP were present or absent. The active fragment exhibited Ca2+/calmodulin-dependent kinase activity with kinetic parameters identical with those of the activated holoenzyme. The key autophosphorylation site of CaM kinase II was absent from the active fragment which indicates that proteolysis can effectively uncouple the activation state and Ca2+/calmodulin independence of the kinase from the action of phosphoprotein phosphatases. Because autophosphorylation exerts such a tight control over this irreversible process, proteolytic activation of CaM kinase II by intracellular proteases offers an attractive mechanism for prolonging the effects of Ca2+ at the synapse.     

Proteolytic cleavage of an activated subcomponent of the first component of rabbit complement, C1s

When rabbit C1 purified by affinity chromatography on IgG-Sepharose 6B was chromatographed on DEAE-cellulose in the presence of ethylenediaminetetraacetate, C1s was isolated as two forms, C1s(I) and C1s(II), having different molecular weights. On the other hand, incubation of the C1 with soybean trypsin inhibitor before the chromatography resulted in the isolation of C1s(I) alone, indicating that, during the purification, C1s(II) was derived from C1s(I) by proteolytic cleavage of C1s(I) by a contaminating protease, probably plasmin [EC 3.4.21.7]. In fact, C1s(I) was completely converted to C1s(II) or a C1s(II)-like fragment by highly purified plasmin. Analysis of the polypeptide chain structures revealed that C1s(I), which consisted of H and L chains with molecular weights of 70,000 and 36,000, respectively, was converted to C1s(II) by cleavage of the H chain, since C1s(II) consisted of two chains each with a molecular weight of 37,000. This conversion proceeded without any alteration in C1 esterase activity, but was accompanied by loss of the ability to form C1r-C1s complex.     

Purification and partial characterization of membrane-associated type II (cGMP-activatable) cyclic nucleotide phosphodiesterase from rabbit brain

Membrane-associated, Type II (cGMP-activatable) cyclic nucleotide phosphodiesterase (PDE) from rabbit brain, representing 75% of the total homogenate Type II PDE activity, was purified to apparent homogeneity. The enzyme was released from 13,000 x g particulate fractions by limited proteolysis with trypsin and fractionated using DE-52 anion-exchange, cGMP-Sepharose affinity and hydroxylapatite chromatographies. The enzyme showed 105 kDa subunits by SDS-PAGE and had a Stokes radius of 62.70 A as determined by gel filtration chromatography. Hydrolysis of cAMP or cGMP showed positive cooperativity, with cAMP kinetic behavior linearized in the presence of 2 microM cGMP. Substrate concentrations required for half maximum velocity were 28 microM for cAMP and 16 microM for cGMP. Maximum velocities were approx. 160 mumol/min per mg for both nucleotides. The apparent Kact for cGMP stimulation of cAMP hydrolysis at 5 microM substrate was 0.35 microM and maximal stimulation (3-5-fold) was achieved with 2 microM cGMP. Cyclic nucleotide hydrolysis was not enhanced by calcium/calmodulin. The purified enzyme can be labeled by cAMP-dependent protein kinase as demonstrated by the incorporation of 32P from [gamma-32P]ATP into the 105 kDa enzyme subunit. Initial experiments showed that phosphorylation of the enzyme did not significantly alter enzyme activity measured at 5 microM [3H]cAMP in the absence or presence of 2 microM cGMP or at 40 microM [3H]cGMP. Monoclonal antibodies produced against Type II PDE immunoprecipitate enzyme activity, 105 kDa protein and 32P-labeled enzyme. The 105 kDa protein was also photoaffinity labeled with [32P]cGMP. The purified Type II PDE described here is physicochemically very similar to the isozyme purified from the cytosolic fraction of several bovine tissues with the exception that it is predominantly a particulate enzyme. This difference may reflect an important regulatory mechanism governing the metabolism of cyclic nucleotides in the central nervous system.     

Oxidative stress employs phosphatidyl inositol 3-kinase and ERK signalling pathways to activate cAMP phosphodiesterase-4D3 (PDE4D3) through multi-site phosphorylation at Ser239 and Ser579

RAW macrophages, which express the PDE4D3 and PDE4D5 cAMP phosphodiesterase isoforms, exhibited increased PDE4 activity when challenged with H2O2 in a fashion that was negated by treatment with the cell permeant antioxidant, N-acetyl cysteine and by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. In Cos1 cells transfected to express PDE4D3, challenge with H2O2 caused a rapid increase in both the activity and phosphorylation of PDE4D3. Lysates from H2O2-treated COS cells caused the phosphorylation of purified, recombinant PDE4D3 at two sites. One was the established ERK phosphorylation site at Ser579, located at the extreme C-terminus of the catalytic unit, and the other was a novel site at Ser239, located at the extreme N-terminus of the catalytic unit. Double Ser239Ala:Ser579Ala mutation of PDE4D3 prevented its H2O2-dependent phosphorylation both in vitro and in intact COS cells. Phosphorylation of PDE4D3 at Ser579 was ablated by treating COS cells with the MEK inhibitor, PD98059, which also negated activation. The activity of the Ser239Ala:Ser579Ala double mutant, and the Ser579Ala single PDE4D3 mutant was unaffected by H2O2 challenge of COS cells, whilst the Ser239Ala mutant was inhibited. Wortmannin inhibited the H2O2-dependent phosphorylation of PDE4D3 in COS cells by around 50%, whilst it fully ablated phosphorylation at Ser239 as well as ablating activation of PDE4D3. Neither immunodepletion of p70S6 kinase nor siRNA-mediated knockdown of mTor inhibited the H2O2-dependent phosphorylation of PDE4D3 at Ser239. Activation of PDE4D3 by challenge with H2O2 was not additive with activation through protein kinase A (PKA)-mediated phosphorylation of PDE4D3. Challenge with H2O2 did not alter PKA-mediated phosphorylation of PDE4D3 at Ser54. H2O2 dependent phosphorylation of PDE4D3, at Ser239 and Ser579, did not alter the sensitivity of PDE4D3 to inhibition by the selective PDE4 inhibitor, rolipram. An unknown protein kinase acting downstream of phosphatidyl inositol 3-kinase phosphorylates PDE4D3 at Ser239. This switches the effect of phosphorylation by ERK at Ser579 from inhibition to activation. We propose that phosphorylation at Ser239 attenuates interaction between either UCR2 or the UCR1/UCR2 module and the PDE4 catalytic unit so as to re-programme the functional outcome effect of phosphorylation by ERK. We identify a novel process through which reactive oxygen species activate long PDE4 isoforms so as to reduce cAMP levels and thereby promote inflammatory responses.     

Proteolytic conversion of xanthine dehydrogenase to xanthine oxidase: evidence against a role for calcium-activated protease (calpain)

The present study tested the hypothesis that calpain is responsible for the limited proteolytic conversion of xanthine dehydrogenase (XD) to xanthine oxidase (XO). We compared the effects of various proteases on the activity and molecular weight of a purified preparation of xanthine dehydrogenase from rat liver. In agreement with previous reports, trypsin treatment produced a complete conversion of XD to XO accompanied by a limited proteolysis of XDH from an Mr of 140 kD to an Mr of 90 kD. Treatment with calpain I or calpain II did not produce a conversion from XD to XO nor did it result in partial proteolysis of the enzyme. Similarly, trypsin treatment partially degraded a reversibly oxidized form of xanthine dehydrogenase while calpain I or calpain II were ineffective. The possibility that an endogenous inhibitor prevented the proteolysis of XDH by calpain I or II was excluded by verifying that brain spectrin, a known calpain substrate, was degraded under the same incubation conditions. The results indicate that calpain is not likely to be responsible for the in vivo conversion of XD to XO under pathological conditions.     

Adenosine 3'':5''-cyclic monophosphate-dependence of protein kinase isoenzymes from mouse liver.

Conditions influencing the cyclic AMP-dependence of protein kinase (ATP-protein phosphotransferase, EC 2.7.1.37) during the phosphorylation of histone were studied. Protein kinase from mouse liver cytosol and the two isoenzymes [PK (protein kinase) I and PK II] isolated from the cytosol by DEAE-cellulose chromatography were tested. A relation between concentration of enzyme and cyclic AMP-dependence was observed for both isoenzymes. Moderate dilution of isoenzyme PK II decreased the stimulation of the enzyme by cyclic AMP. Isoenzyme PK I could be diluted 200 times more than isoenzyme PK II before the same decrease in cyclic AMP-dependence appeared. Long-term incubation with high concentrations of histone increased the activity in the absence of cyclic AMP relative to the activity in the presence of the nucleotide. This was more pronounced for isoenzyme PK II than for isoenzyme PK I. The cyclic AMP concentration needed to give half-maximal binding of the nucleotide was the same as the cyclic AMP concentration (Ka) at which the protein kinase had 50% of its maximal activity. The close correlation between binding and activation is also found in the presence of KCl, which increased the apparent activation constant (Ka) for cyclic AMP. With increasing [KCl], a progressively higher proportion of the histone phosphorylation observed in cytosol was due to cyclic AMP-independent (casein) kinases, leading to an overestimation of the degree of activation of the cyclic AMP-dependent protein kinases present. The relative contributions of cyclic AMP-dependent and -independent kinases to histone phosphorylation at different ionic strengths was determined by use of heat-stable inhibitor and phospho-cellulose chromatography.     

Regulation by insulin of cyclic nucleotide phosphodiesterase (PDE) from rat luteal cells

The effect of insulin on cyclic nucleotide phosphodiesterase (PDE) in rat luteal cells was studied. Cells were obtained from PMSG/hCG primed rats and further incubated or not with insulin. The hormone produced an increase of enzyme activity after a 10 min incubation of intact cells. Maximal stimulation was achieved at 0.2 nM of insulin. Two peaks of cyclic nucleotide phosphodiesterase activity were resolved after chromatography of cell cytosolic extracts on DEAE-cellulose. These peaks (I and II) were active with cAMP as substrate but only peak I was active with cGMP. The enzyme activity of both peaks was increased in cells treated with insulin. Phosphodiesterase activity in the two peaks show two kinetic components for cAMP hydrolysis, one of high affinity (Km 2-4 microM) and the other of low affinity (47-56 microM). Treatment of the cells with insulin produced a 2 to 8 fold increase of the Vmax of these peaks. In addition after stimulation with insulin, the activation of peak I phosphodiesterase by calmodulin was less effective.     

Regulation of cyclic adenosine 3':5'-monophosphate phosphodiesterases: altered pattern in transformed myoblasts

Rat skeletal myoblasts, L6 and L8, have two major forms of phosphodiesterases, PDE II and PDE III. Only the former is activated by treatment with proteases. When the myoblasts are exposed to cAMP for 10-16 h, the activity of PDE III increases considerably. This increase is accompanied by a loss of activatability of PDE II by proteases. Leupeptin prevents the increase in the levels of PDE III suggesting that a protease in vivo may be responsible for the formation of PDE III from PDE II. Spontaneously or Rous sarcoma virus-transformed myoblasts, however, show altered regulation of the two forms of PDE. In the presence of cAMP in the medium, unlike the nontransformed cells, the levels of PDE III do not increase but the activity of PDE II rises. Simultaneously, PDE II becomes refractory to activation by proteases. The altered mode of PDE regulation in transformed cells is dominant in hybrids between normal and transformed myoblasts, which suggests that altered regulation is due to an "acquisition" of some new property by transformed cells.     

Protease II from Escherichia coli. Purification and characterization.

We have previously demonstrated the existence of two types of endopeptidase in Escherichia coli. A purification procedure is described for one of these, designated protease II. It has been purified about 13,500-fold with a recovery of 24%. The isolated enzyme appears homogeneous by electrophoresis and gel filtration. Its molecular weight is estimated by three different methods to be about 58,000. Its optimal pH is around 8. Protease II activity is unaffected by chelating agents and sulfhydryl reagents. Amidase and proteolytic activities are stimulated by calcium ion, which decreases the enzyme stability. Like pancreatic trypsin, this endopeptidase catalyses the hydrolysis of alpha-amino-substituted lysine and arginine esters. It appears distinct from the previously isolated protease I, which is a chymotrypsin-like enzyme. The apparent Michaelis constant for hydrolysis of N-benzoyl-L-arginine ethyl ester is 4.7 X 10(-4) M. The esterase activity is inhibited by diisopryopylphosphorofluoridate (Ki(app) equals 2.7 X 10(-3) M) and tosyl lysine chloromethyl ketone (Ki(app) equals 1.8 X 10(-5) M), indicating that serine and histidine residues may be present in the active site. However, protease II is insensitive to phenylmethanesulfonyl fluoride and several natural trypsin inhibitors. Its amidase and esterase activities are competitively inhibited by free arginine and aromatic amidines. The proteolytic activity measured on axocasein is very low. In contrast to trypsin, protease II is without effect on native beta-galactosidase. It easily degrades aspartokinase I and III. Nevertheless both enzymes are resistant to proteolysis in the presence of their respective allosteric effectors. These results provide further evidence that such differences in protease susceptibility can be related to the conformational state of the substrate. The possible implication of structural changes in the mechanism of preferential proteolysis in vivo, is discussed.     

Phosphorylation-mediated activation and translocation of the cyclic AMP-specific phosphodiesterase PDE4D3 by cyclic AMP-dependent protein kinase and mitogen-activated protein kinases. A potential mechanism allowing for the coordinated regulation of PDE4D activity and targeting

In this study, we describe a novel mechanism by which a protein kinase C (PKC)-mediated activation of the Raf-extracellular signal-regulated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) cascade regulates the activity and membrane targeting of members of the cyclic AMP-specific phosphodiesterase D family (PDE4D). Using a combination of pharmacological and biochemical approaches, we show that increases in intracellular cAMP cause a protein kinase A-mediated phosphorylation and activation of the two PDE4D variants expressed in vascular smooth muscle cells, namely PDE4D3 and PDE4D5. In addition, we show that stimulation of PKC via the associated activation of the Raf-MEK-ERK cascade results in the phosphorylation and activation of PDE4D3 in these cells. Furthermore, our studies demonstrate that simultaneous activation of both the protein kinase A and PKC-Raf-MEK-ERK pathways allows for a coordinated activation of PDE4D3 and for the translocation of the particulate PDE4D3 to the cytosolic fraction of these cells. These data are presented and discussed in the context of the activation of the Raf-MEK-ERK cascade acting to modulate the activation and subcellular targeting of PDE4D gene products mediated by cAMP.