Plasma Membrane Protein Kinase Research Articles

TMK4 receptor kinase negatively modulates ABA signaling by phosphorylating ABI2 and enhancing its activity.

In plants, clade A type 2C protein phosphatases (PP2CAs) have emerged as major players in abscisic acid (ABA)-regulated stress responses by inhibiting protein kinase activity. However, how different internal and external environmental signals modulate the activity of PP2CAs are not well known. The transmembrane kinase (TMK) protein 4 (TMK4), one member of a previously identified receptor kinase subfamily on the plasma membrane that plays vital roles in plant cell growth, directly interacts with PP2CAs member (ABA-Insensitive 2, ABI2). tmk4 mutant is hypersensitive to ABA in both ABA-inhibited seed germination and primary root growth, indicating that TMK4 is a negative regulator in ABA signaling pathway. Further analyses indicate that TMK4 phosphorylates ABI2 at three conserved Ser residues, thus enhancing the activity of ABI2. The phosphorylation-mimic ABI2S139DS140DS266D can complement but non-phosphorylated form ABI2S139AS140AS266A cannot complement ABA hypersensitive phenotype of the loss-of-function mutant abi1-2abi2-2. This study provides a previously unidentified mechanism for positively regulating ABI2 by a plasma membrane protein kinase.

Cold Signal Shuttles from Membrane to Nucleus

In this issue of Molecular Cell, Liu etal. (2017) show that the cold-activated plasma membrane protein kinase CRPK1 phosphorylates 14-3-3 proteins, triggering its nuclear translocation to impair the stabilization of thetranscription factor CBFs for a feedback excessive cold defense response during the freezing in Arabidopsis.

FRET biosensors reveal AKAP‐mediated shaping of subcellular PKA activity and a novel mode of Ca2+/PKA crosstalk

A‐Kinase Anchoring Proteins (AKAPs) serve as scaffolds for cAMP‐dependent Protein Kinase (PKA) and other signaling enzymes within specific subcellular compartments. However, some AKAPs are not static anchors, but form dynamic signaling complexes that can traffic to different subcellular compartments when triggered. Gravin (AKAP12) is a multivalent AKAP that anchors PKA and other enzymes to the plasma membrane but redistributes to the cytosol upon intracellular Ca2+ elevation. Through the redistribution of gravin, we postulate that Ca2+ may regulate the interaction of PKA with downstream substrates, thus representing a novel crosstalk mechanism between Ca2+ and PKA‐dependent pathways. To assess this, we measured the impact of gravin‐V5/His expression on compartmentalized PKA activity using the PKA FRET biosensor AKAR3. In cultured AN3 CA cells, which lack endogenous gravin, expression of gravin‐V5/His increased forskolin‐stimulated PKA activity in AKAR3 constructs targeted to the plasma membrane compared to control cells lacking gravin or expressing a ΔPKA‐gravin construct missing the PKA‐binding domain. In contrast, gravin‐V5/His expression decreased forskolin‐stimulated PKA activity in cytosolic AKAR3 constructs. A mutant gravin construct with inhibited membrane localization had no effect on membrane and cytosolic PKA activity. Finally, pretreatment with the calcium‐elevating agent thapsigargin caused the redistribution of gravin and prevented the gravin‐mediated increase in plasma membrane PKA activity following forskolin or isoproterenol treatment. These results reveal that Ca2+ alters receptor‐mediated PKA activity through gravin redistribution and supports the hypothesis that gravin mediates crosstalk between Ca2+ and PKA‐dependent pathways.

Changes in Rat Myometrial Plasma Membrane Protein Kinase A Are Confined to Parturition

We have previously shown that pregnant rat myometrial plasma membrane-associated cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) decreases prior to delivery, coincident with a decline in the inhibitory effect of cAMP on contractant-stimulated parameters. We now find that rat myometrial membrane-associated PKA concentrations in early to mid-pregnancy are equivalent to those in cycling rats. Following the decline associated with parturition, membrane PKA recovers within 1 to 2 days postpartum. Treatment with the antiprogestin onapristone caused a decrease in myometrial membrane PKA catalytic and regulatory subunits compared to untreated controls by 12 hours. This coincided temporally with recently reported increases in electrical and contractile activity. In unilaterally pregnant rats, the decline in plasma membrane PKA was observed in both nonpregnant and pregnant horns but was more rapid in the pregnant horns. These data indicate that the myometrial plasma membrane PKA pattern before and during most of pregnancy is not consistent with progesterone exerting a primary influence on PKA membrane localization. Rather, the fall in membrane PKA associated with parturition may contribute to or be influenced by the increased contractile and electrical activity of labor that is a consequence of the loss of progesterone influence and is not absolutely dependent on the presence of fetuses.

Scar Formation

Injured tissue produces fibrin, which makes a provisional extracellular matrix (ECM). Degradation of this fibrin ECM by plasmin is required for minimizing scar formation. Plasmin is produced by proteolytic cleavage of plasminogen by plasminogen activators (tPA and uPA), which are inhibited by plasminogen activator inhibitor-1 (PAI-1). Sachs et al . find that mice deficient in p75 neurotrophin receptor (p75 NTR ) exhibit reduced fibrin deposition after sciatic nerve crush injury or lipopolysaccharide-induced pulmonary fibrosis. The decrease in fibrin associated with sciatic nerve injury was due to increased expression and activity of tPA, whereas the decrease in fibrin associated with pulmonary fibrosis was due to decreased PAI-1 abundance. In wild-type mice after sciatic nerve injury, Schwann cells in which p75 NTR was abundant lacked tPA, which suggests that cells expressing p75 NTR did not express tPA. The increased proteolytic activity toward fibrin (measured by in situ zymography) associated with the nerve injury in p75 NTR −/− mice was prevented by application of a specific inhibitor of tPA or by crossing the mice with tPA −/− mice to produce mice deficient in both tPA and p75 NTR . When cultured on a fibrin gel, Schwann cells from the p75 NTR −/− mice degrade the fibrin, whereas wild-type Schwann cells form a monolayer. Studies with a transfected fibroblast cell line overexpressing p75 NTR showed increased abundance of transcripts for PAI-1 and decreased transcripts and activity of tPA. Transcriptional regulation of tPA required a cAMP (adenosine 3′,5′-monophosphate) pathway. Inhibition of tPA activity in the p75 NTR -overexpressing cells was prevented if cAMP concentrations were increased pharmacologically and cAMP concentrations were decreased in the p75 NTR -overexpressing cells. The injured sciatic nerves also showed increased cAMP in the p75 NTR −/− mice compared with injured nerves from wild-type mice. Coimmunoprecipitation studies and in vitro interaction studies indicated that p75 NTR and phosphodiesterase 4A5 (PDE4A5) interacted. The authors propose that recruitment of PDE4A5 to the membrane inhibited cAMP signaling, a hypothesis supported by decreased activation of a plasma membrane protein kinase A reporter in the p75 NTR -overexpressing cells. Tissue remodeling and scar formation appear to be additional ways that p75 NTR signaling is involved in response to injury. B. D. Sachs, G. S. Baillie, J. R. McCall, M. A. Passino, C. Schachtrup, D. A. Wallace, A. J. Dunlop, K. F. MacKenzie, E. Klussmann, M. J. Lynch, S. L. Sikorski, T. Nuriel, I. Tsigelny, J. Zhang, M. D. Houslay, M. V. Chao, K. Akassoglou, p75 neurotrophin receptor regulates tissue fibrosis through inhibition of plasminogen activation via a PDE4/cAMP/PKA pathway. J. Cell Biol. 177 , 1119-1132 (2007). [Abstract] [Full Text]

Progesterone prevents the pregnancy-related decline in protein kinase A association with rat myometrial plasma membrane and A-kinase anchoring protein.

The presence of cAMP-dependent protein kinase (PKA) in the plasma membrane compartment and its association with an A-kinase anchoring protein (AKAP150) is implicated in mediating cAMP regulatory events in the rat myometrium. The association of PKA with purified myometrial plasma membrane declined gradually between Day 16 and Day 21 of gestation, with a decrease of 53% +/- 11% of the catalytic subunit and of 61% +/- 7% of the regulatory subunit at Day 21 compared with Day 19. To determine the role of progesterone in this association, pregnancy was prolonged by administration of progesterone or shortened by administration of the antiprogestin RU486. Progesterone treatment maintained PKA association with plasma membrane at Day 21 at 123% +/- 23% (catalytic subunit) and 92% +/- 4% (regulatory subunit) of Day 19 levels. In contrast, protein phosphatase 1, protein phosphatase 2B, phospholipase Cbeta(3), and AKAP150 concentrations in the plasma membrane did not change over this interval or with progesterone treatment. Changes in PKA coimmunoprecipitated with membrane-associated AKAP150 paralleled those in total plasma membrane on Days 19 and 21 and on Day 21 following progesterone treatment. In contrast, plasma membrane PKA catalytic and regulatory subunits decreased by 20 h after RU486 injection on Day 15 of pregnancy to levels resembling those on Day 21. These data indicate that progesterone prevents the decline in PKA associated with myometrial plasma membrane and with AKAP150 in the pregnant rat. The decrease in membrane-bound PKA between Days 19 and 21 and after RU486 treatment precedes the onset of parturition in both experimental paradigms. The loss of plasma membrane PKA may be critical for the decrease in the inhibitory effect of cAMP on oxytocin-induced phosphatidylinositide turnover that occurs near the end of pregnancy and may contribute to enhanced myometrial contractile responsiveness near term.

Inhibition of insulin-stimulated phosphorylation of the intracellular domain of phospholemman decreases insulin-dependent GLUT4 translocation in streptolysin-O-permeabilized adipocytes

A variety of studies indicate that protein kinase C might be involved in the insulin signalling cascade leading to translocation of the insulin-regulated glucose transporter GLUT4 from intracellular pools to the plasma membrane. Phospholemman is a plasma-membrane protein kinase C substrate whose phosphorylation is increased by insulin in intact muscle [Walaas, Czernik, Olstad, Sletten and Walaas (1994) Biochem. J. 304, 635-640]. The present study examined whether the inhibition of phospholemman phosphorylation modulates the effects of insulin on GLUT4 translocation. For this purpose, a synthetic peptide derived from the intracellular domain of phospholemman with the phosphorylatable serine residues replaced with alanine residues was prepared. This peptide was found to decrease the protein kinase C-catalysed phosphorylation of a synthetic phospholemman peptide in vitro. When introduced into streptolysin-O-permeabilized adipocytes, the peptide decreased the effects of insulin on both the phosphorylation of phospholemman and the recruitment of GLUT4 to the plasma membrane. Similarly, the internalization of phospholemman antibodies, which also decreased the protein kinase C-mediated phosphorylation of the synthetic phospholemman peptide in vitro, decreased the effect of insulin on GLUT4 translocation in the adipocytes. The results suggest that phosphorylation of the intracellular domain of phospholemman might be involved in modulating the insulin-induced translocation of GLUT4 to the plasma membrane.

DUNALIELLA—A MODEL EXTREMOPHILIC ALGA

The unicellular algaDunaliellais unique in its ability to adapt to extreme environmental conditions. Adaptation to extreme salinity involves short-term and long-term responses. The former include osmotic adjustment by accumulation of large amounts of intracellular glycerol and efficient elimination of Na+ions by plasma membrane transporters. The latter involves synthesis of two extrinsic plasma membrane proteins: a carbonic anhydrase and a novel type of a transferrin-like protein. These proteins are associated with acquisition of CO2and Fe, respectively, whose availability is diminished in high salinity, limiting algal growth. Both proteins are functional over a wide range of salt concentrations and differ in structure from their mesophilic counterparts in possessing additional internal repeats and in having higher ratios of acidic: basic amino acids.Dunaliella acidophilasurvives at pH 0–1 by overexpression of a potent plasma membrane H+-ATPase which provides effective capacity for elimination of protons. Sequence comparisons of the ATPase genes from halophilic and acidophilic species reveals variations in charged amino acid composition within a distinct extrinsic C-terminal domain of the protein.Dunaliella bardawiladapts to high light intensity by several strategies: it accumulates large amounts of β-carotene which screens the photosynthetic system against photoinhibition and it modifies the photosynthetic machinery by synthesis of a special light-harvesting protein which presumably functions in dissipation of excessive light energy. Both responses depend on synthesis of special proteins and enzymes. Signal transduction mechanisms mediating stress responses inDunaliellaare poorly understood. Sensing osmotic/salinity changes involves specific plasma membrane sterols and activation of a plasma membrane protein kinase. Induction of β-carotene accumulation can be mimicked by reactive oxygen species generators.

Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C.

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)2D3 and 24,25-(OH)2D3. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1 alpha, 25-(OH)2D3 (1,25) and 24R,25-(OH)2D3 (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1, 25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKC alpha and zeta as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [gamma 32 P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A1 (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25 but not 1, 25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase.

Vitamin D3 regulation of stromelysin‐1 (MMP‐3) in chondrocyte cultures is mediated by protein kinase C

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)2D3 and 24,25-(OH)2D3. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1α,25-(OH)2D3 (1,25) and 24R,25-(OH)2D3 (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1,25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKCα and ζ as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [γ32P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A, (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25, but not 1,25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase. © 1996 Wiley-Liss, Inc.

A novel chloride-dependent GTP-utilizing protein kinase in plasma membranes from human respiratory epithelium

The protein kinases that stimulate ion flux across airway epithelium are believed to utilize ATP as phosphate donor. Here we show that a chloride-sensitive protein kinase (in an apically enriched plasma membrane fraction from human nasal respiratory epithelium) uses guanosine 5'-triphosphate in preference to ATP as phosphate donor and is not inhibited by the protein kinase inhibitors staurosporine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, and N-(2-guanodinoethyl)-5-isoquinoline sulfonamide. This kinase phosphorylates a 37-kDa membrane protein (p37), which exhibits a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive phosphorylation peak at 40 mM Cl- (DIDS inhibition constant = 8 microM). p37 is additionally phosphorylated by an N-(2-guanodinoethyl)-5-isoquinoline sulfonamide-inhibitable protein kinase that uses ATP and shows a similar chloride sensitivity. The profile of membrane phosphoproteins generated by both kinases is also dependent on the source of Pi, the species of anion, and the concentration of anion. We propose a molecular mechanism for the transduction of Cl- concentration into a guanosine 5'-triphosphate-selective protein kinase signal and show that anion substitution alters the intensity of phosphorylation of membrane proteins in the absence of exogenously added protein kinases.

Accumulation of 1,2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats.

Choline deficiency, via deprivation of labile methyl groups, is associated with a greatly increased incidence of hepatocarcinoma in experimental animals. This dietary deficiency also causes fatty liver, because choline is needed for hepatic secretion of lipoproteins. We hypothesized that fatty liver might be associated with the accumulation of 1,2-sn-diradylglycerol and subsequent activation of protein kinase C. Several lines of evidence indicate that cancers might develop secondary to abnormalities in protein kinase C-mediated signal transduction. We observed that rats fed a choline-deficient diet for 1, 6, or 27 weeks had increased hepatic concentrations of 1,2-diradylglycerol. At 1 and 6 weeks, hepatic plasma membrane from choline-deficient rats had increased concentrations of 1,2-sn-diacylglycerol and 1-alkyl, 2-acylglycerol, with the latter accounting for 20-26% of membrane 1,2-sn-diradylglycerol (as compared with only 2-5% in controls). Protein kinase C activity was increased in hepatic plasma membrane at 1 week of choline deficiency. By Western blotting there was an increase in the amount of protein kinase C zeta and a decrease in the amount of protein kinase C delta in liver at 1 week. By 6 weeks of choline deficiency, hepatic plasma membrane and cytosolic protein kinase C (PKC) activities were increased significantly, with increased amounts of hepatic plasma membrane protein kinase C alpha, and delta detected by Western blotting. Glycogen synthase activity in liver was diminished after 1 week of choline deficiency; this enzyme is inhibited by PKC-mediated phosphorylation. We suggest that choline deficiency perturbed PKC-mediated transmembrane signaling within liver and that this contributed to the development of hepatic cancer in these animals.

Phospholipid-dependence of rat liver plasma membrane protein kinase activities — a new approach

The influence of the phospholipid composition and fluidity on protein kinase A and protein kinase C activities in rat liver plasma membrane was studied. We observed that enrichment of membranes with phosphatidylglycerol, phosphatidylserine, phosphatidylethanolamine and dioleoylphosphatidylcholine caused activation of both protein kinases. Phosphatidylglycerol was found to be most effective activator. The enrichment of plasma membranes with dipalmitoylphosphatidylcholine and sphingomyelin led to decrease in protein kinase A and C activities. The stimulatory effect of phosphatidylglycerol was confirmed in plasma membranes pretreated with exogenous phospholipases A 2, C and D, and subsequently enriched with phosphatidylglycerol. We suggest that besides the specific presence of definite phospholipids protein kinases A and C require a more fluid membrane lipid bilayer to display an optimal activity.

Endogenous activity of cyclic nucleotide-dependent protein kinase in plasma membranes isolated from Strongylocentrotus purpuratus sea urchin sperm

Activity of cyclic nucleotide-dependent protein kinase was investigated in flagellar plasma membranes of sea urchin sperm (S. purpuratus). Membranes incubated with [gamma-32P]ATP showed in the presence of 1 microM cAMP an increased phosphorylation in multiple polypeptides. Half maximal response was seen at 0.6 microM of cAMP. In contrast, higher concentrations (100 microM) of cGMP were required to cause the same amount of protein phosphorylation. 80% of the protein kinase activity stimulatable by cAMP was resistant to extraction by 10 mM EGTA and sonication but it was entirely recovered in a detergent-solubilized fraction. Membranes pretreated with 200 microM cAMP, ultracentrifuged and resuspended in buffer solution did not undergo cAMP-stimulated phosphorylation in their polypeptides. This study demonstrates that flagellar plasma membranes isolated from S. purpuratus sea urchin sperm have an endogenous cAMP-dependent protein kinase, which may be bound to the membrane via its regulatory subunit.

Properties of enzyme activities involved in protein phosphorylatino-dephosphorylation of the thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca 2+-dependent protien kinase activities as well as a basal (cAMP- and Ca 2+-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent K m values of cAMP and Ca 2+ for the membrane-bound protein kinase were 5·10 −8 M and 8.3·10 −4M (in the presence of 1 mM EGTA), respectively. The apparent K m values of Mg 2+ were 7·10 −4 M (without cAMP and Ca 2+, 5·10 −4 M (with cAMP) and 1.3·10 −3 M (with Ca 2+), and those ATP were 3.5·10 −5 M (with or without cAMP) and 8.5·10 −5 M (with Ca 2+). The Ca 2+-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using 32P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca 2+, but was sitmulated by a rather broad range (5–25 mM) of Mg 2+ and Mn 2+. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca 2+-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.

ラット正常肝細胞と肝がん細胞の細胞膜生理活性の差異

The cancerous changes of plasma membrane activities were reviewed with special reference to the controlling mechanisms of cell proliferation. The article includes the following chapters in addition to introduction and conclusion. Chapter I. Cancerous changes of enzyme systems forming cyclic nucleotides; (a) adenylate cyclase and (b) guanylate cyclase. Chapter II. Cancerous change of cAMP-dependent protein kinase in plasma membranes. Chapter III. Role of plasma membranes in controlling the intracellular Ca2+ concentration, and cancerous changes of the controlling systems. Chapter IV. Cancerous changes of Ca2+calmodulin-dependent regulatory systems in plasma membranes; (a) calmodulin, (b) cAMP phosphodiesterase and (c) Ca2+, Mg2+-ATPase. Chapter V. Cancerous changes of other marker enzymes in plasma membranes; (a) 5'-nucleotidase, (b) alkaline phosphatase, and (c) ATPase.

A protein kinase of the plasma membrane of Dictyostelium discoideum

D. discoideum amoebae were found to phosphorylate plasma membrane proteins when intact cells were incubated with either [γ- 32P]ATP or [ 32P]phosphate. In the first case, the incorporation was largely a consequence of the hydrolysis of [γ- 32P]ATP, cellular uptake of the generated [ 32P]phosphate and its subsequent incorporation into ATP. When the contribution of this process to the phosphorylating activity of intact cells was eliminated, an ecto-protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity could be demonstrated. As amoebae progressed through their aggregation program, they showed a decreased ability to phosphorylate their plasma membrane when incubated with [γ- 32P]ATP or [ 32P]phosphate. Analysis of ATPase activity, permeability properties and the pattern of proteins phosphorylated by intact cells and isolated plasma membranes lead to the following conclusions: the lower levels of phosphorylation observed with starved cells reflected an altered uptake of [ 32P]phosphate by these cells rather than a significant change in the plasma membrane protein kinase activity. Neither the substrates nor the activity of the ecto-protein kinase was dramatically altered during starvation.

Protein phosphorylation in human peripheral blood lymphocytes. Subcellular distribution and partial characterization of adenosine 3′:5′-cyclic monophosphate-dependent protein kinase

Cytoplasmic and membrane fractions prepared from human peripheral-blood lymphocytes both contained cyclic AMP-dependent protein kinase activity and endogenous protein kinase substrates. Protein kinase activity in the particulate fractions was not eluted with 0.25 M-NaCl, suggesting that it was not derived from non-specifically absorbed soluble cytoplasmic protein kinase. Nor was the particulate protein kinase activity eluted by treatment with cyclic AMP, suggesting that the catalytic subunit is membrane-bound and arguing against cyclic AMP-induced translocation of particulate activity. Cyclic AMP-dependent protein-phosphorylating activity in the cytoplasmic fraction was highly sensitive to inhibition by Mn2+, and was co-eluted from DEAE-cellulose primarily with type-I rabbit skeletal-muscle kinase. Cyclic AMP-dependent phosphorylating activity in the plasma-membrane fractions was stimulated at low [Mn2+] and inhibited only at high [Mn2+]. When solubilized with Nonidet P-40, plasma-membrane protein kinase was co-eluted from DEAE-cellulose with type-II rabbit muscle kinase. These differences, together with the strong association of the particulate kinases with the particulate fraction, suggest the possibility of compartmentalized protein phosphorylation in intact lymphocytes.

A GTP-specific protein kinase in plasma membranes of mouse fibroblasts

Endogenous protein phosphorylation in plasma membranes isolated from SV 40-transformed mouse fibroblasts was studied in the presence of [γ- 32P]GTP. The phosphoprotein pattern showed one main component with 100 k which could not be phosphorylated with ATP. Optimal phosphorylation of pp 100 was dependent on protein concentration. Strict substrate specificity of the corresponding endogenous kinase could be demonstrated by comparative electrophoresis with a plasma membrane sample phosphorylated with [γ- 32P]ATP and by isotope dilution experiments. GTP-specific phosphorylation of pp 100 was not dependent on cyclic nucleotides but influenced by GDP, GMP and AMP.

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [γ- 32P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg 2+ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.