Basic fibroblast growth factor is a substrate for phosphorylation by human neutrophil ecto-protein kinase activity.
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Amino Acids↗
Biomedical subjects
S A Goueli
Publications and source records attributed to S A Goueli.
A procedure for rapid isolation of cloned cDNA inserts using the polymerase chain reaction technique.
A reproducible and rapid procedure for isolation of cloned cDNA insert from a lambda gt11 cDNA library is described. The procedure relies on the polymerase chain reaction method using forward and reverse primers for lambda gt11, followed by isolation of the cloned cDNA insert by a rapid technique. The procedure should also be applicable to isolation of cDNA inserts cloned in other vectors such as lambda gt10.
Animals↗
Differential regulation of prostatic protein kinase C isozymes by androgens.
Multiple isozymes of Ca2+/phospholipid-dependent protein kinase (PKC) were isolated from the rat ventral prostate. The enzyme exists mainly as type II (beta), and type III (alpha) forms, and it is possible that type II isozyme may comprise the subspecies beta 1 and beta 2. The total and specific activities of prostatic PKC isoforms were reduced in castrated animals; this decrease was specific since administration of androgens to castrated animals reversed such a decline. Also, there was a differential response to androgen deprivation so that type III isozyme declined at a faster rate than that of type II. Thus, our studies show for the first time that PKC of the rat ventral prostate comprises multiple isozymes, and that the activity of these various forms are differentially regulated by androgens.
Androgens↗
Tissue-specific decline in cytosolic casein kinase II of the ventral prostate in aging rats.
An age-associated decline in the activity of cytosolic casein kinase II in the rat ventral prostate was observed. The decrease in specific and total activity of casein kinase II of prostatic cytosol obtained from 12-month old rats compared with that from 3-month old rats was 50% and 70%, respectively. This decrease was tissue specific since no such alteration in enzymic activity was found in liver, lung, or heart. The decline in activity was not due to an increase in the concentration of a casein kinase inhibitor or to altered androgenic status with aging. Rather, the reduction in the activity of the enzyme was commensurate with the decrease in the total concentration of the enzyme protein as determined by an ELISA using anti-casein kinase II antibodies. The activity of casein kinase II in the nuclear fraction of the rat prostate was not altered in aging animals. It may be concluded that the cytosolic form of casein kinase II in the prostate of older animals is regulated at the transcriptional level (but not via the androgen-receptor complex mechanism). An alternative explanation for this observation may relate to altered stability of the enzyme in the prostate of older animals.
Aging↗
Monoclonal antibodies against nuclear casein kinase NII (PK-N2).
A monoclonal antibody was produced against the nuclear casein kinase II (PK-N2) isolated from rat liver. The antibody was of the IgM class, and showed immunoreactivity towards the higher molecular weight subunit (41K Da) of the protein kinase in Western blots. The antibody was equally reactive towards the PK-N2 isolated from rat ventral prostate indicating that it can recognize the enzyme from different tissues of the rat. The antibody also detected the cytosolic casein kinase II (CK-2) suggesting significant similarity of the antigenic domains in the two forms of this protein kinase. No binding was detected with the nuclear or cytosolic casein kinase I (PK-N1 and CK-1). The antibody did not inhibit the enzyme activity or directly precipitate the enzyme, but when coupled to an affinity matrix and cross-linked with dimethylpimelimidate, it was capable of removing nearly all the PK-N2 activity from solution.
Animals↗
Polyclonal antibodies against rat liver cytosolic casein kinase II (CK-2) cross-react with CK-2 from other tissues and nuclear form (PK-N2) of the enzyme.
Casein kinase II (CK-2) is a ubiquitous serine/threonine protein kinase, and is localized to both the cell nucleus and cytoplasm. Despite extensive biochemical similarities in their properties, there is evidence that the two forms of the enzyme exhibit certain distinctions (1). This prompted us to produce antibodies against CK-2, which could be utilized as a possible tool for investigations of the various forms of this enzyme. Specific polyclonal antibodies against the rat liver cytosolic CK-2 were raised in egg yolk of laying hens; the enzyme had repeatedly failed to elicit an immunogenic response in rabbits. The purified polyclonal antibody (egg yolk immunoglobulin, IgY) recognized all three subunits (42, 38, and 28 kDa) of the enzyme in immunoblots. The antibody when bound to a matrix was capable of removing CK-2 from solution, and the bound enzyme could be recovered from the immunoaffinity matrix with 0.1 M diethylamine. The antibody exhibited a high affinity towards CK-2 prepared from cytosol of liver, ventral prostate, and several other rat tissues, but no immunoreactivity was detected towards a number of other protein kinases tested. The subunits of the nuclear form of CK-2 (PK-N2) migrated differently when electrophoresed in parallel in the same gel. However, the antibody did cross-react with the various subunits of PK-N2 suggesting a significant homology in the immunogenic domains in the various subunits of the two forms of the enzyme.
Animals↗
Polyamine-like effects of aminoglycosides on various messenger-independent protein kinase reactions.
Gentamicin and several other aminoglycoside antibiotics in millimolar concentrations directly stimulate the phosphorylation of casein by purified preparations of cAMP- and Ca2+-independent protein kinases PK-C2 (equivalent to cytosolic casein kinase II) and its nuclear counterpart PK-N2 from rat liver and ventral prostate. These stimulatory effects of aminoglycoside antibiotics were similar to those exerted by the aliphatic polyamine spermine. Phosphorylation of casein by purified preparations of messenger-independent protein kinases PK-C1 (equivalent to cytosolic casein kinase I) and its nuclear counterpart PK-N1 was much less enhanced by spermine and the aminoglycoside antibiotics tested. Stimulations of PK-N2 reactions evoked by gentamicin or spermine (at 0.5 and 1.0 mM) were not additive. Several amino sugars tested were without effect on these protein kinases. Methylglyoxal bis(guanylhydrazone) which is known to block the stimulatory effects of polyamines on certain other enzymes did not alter spermine-stimulated phosphorylation of casein catalyzed by PK-N2 preparations.
Aminoglycosides↗
Purification of nuclear cAMP-independent protein kinases from rat ventral prostate.
Two nuclear cAMP-independent protein kinases (designated PK-N1 and PK-N2) were purified from rat ventral-prostate and liver. The yield of enzyme units was 4-5% and 7-9% for each enzyme from the prostatic nuclei and liver nuclei, respectively. The average fold purification for prostatic nuclear protein kinase N1 and N2 was 1360 and 1833, respectively. The respective average specific activity of the two enzymes towards casein was 81,585 and 110,000 nmol 32P incorporated/hr/mg of enzyme. Protein kinase N1 comprised one polypeptide of Mr 35,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase N2 comprised two polypeptides Mr 40,000 and 30,000 of which only the Mr 30,000 polypeptide was autophosphorylated. Both enzymes were active towards casein, phosvitin, dephosphophosvitin, spermine-binding protein, and non-histone proteins in vitro. Little activity was detected towards histones. Both enzymes were stimulated by 150-200 mM NaCl. MgCl2 requirement varied with the protein substrate but was between 2-4 mM for both enzymes. With dephosphophosvitin as substrate, the apparent Km for ATP for N1 protein kinase was 0.01 mM. GTP did not replace ATP in this reaction. Protein kinase N2 was active in the presence of ATP or GTP. The apparent Km was 0.01 mM for ATP, but 0.1 mM for GTP.
Animals↗
Purification of cytosolic cAMP-independent protein kinases from rat ventral prostate.
Two cAMP-independent protein kinases were purified from rat ventral-prostate and liver cytosol, and were designated PK-C1 and PK-C2 to distinguish them from the nuclear protein kinases described in the preceding paper. The yield of the prostate enzymes was about 5% each, and about 10% each for the liver enzymes. The average fold purification of the prostatic enzymes was 1892 and 3176 for protein kinase C1 and C2, respectively. Their average respective specific activity towards casein was 40,111 and 67,340 nmol 32P incorporated/hr per mg of enzyme protein. protein kinase C1 comprised one polypeptide of Mr 39,000 which underwent phosphorylation in the presence of Mg2+ + ATP. Protein kinase C2 comprised three polypeptides of Mr 41,000; 38,000; 26,000. Of these only the Mr 26,000 polypeptide was autophosphorylated. The Mg2+ requirement for protein kinase C1 and C2 was between 1 and 4 mM depending on the nature of the protein substrate. Both enzymes were stimulated by 100-200 mM NaCl. Km for ATP for C1 and C2 kinases was 0.01 mM; GTP could be used only by protein kinase C2 but with a markedly lower affinity. The enzymes were active towards casein, phosvitin, dephosphophosvitin, and spermine-binding protein in vitro, but demonstrated little activity towards histones. Despite several similarities in these general properties of cytosolic protein kinases C1 and C2 with those of nuclear protein kinases N1 and N2, a number of differences are also noted.
Animals↗
Mechanisms and significance of polyamine stimulation of various protein kinase reactions.
An overview of the work on polyamine effects on certain protein kinase reactions is presented. In general, the reactions catalyzed by the messenger-independent protein kinases but not by cyclic nucleotide-, Ca2+-, Ca2+-calmodulin-, and Ca2+-anionic lipid-dependent protein kinases, are markedly enhanced by polyamines. The extent of this stimulation depends critically on the nature of the protein substrate and several other factors. A variety of other polycationic compounds including Co3+(NH3)6, polybrene, and certain aminoglycoside antibiotics exert polyamine-like effects in the same reactions. These observations suggest that the charge properties rather than any strict chemical structure play a role in the action of polyamines. Available data do not support a specific "cofactor" function of these amines for the protein kinases involved in the polyamine-stimulable reactions. It appears that the action of polyamines is mediated via their influence on the conformational status of the protein substrates thereby altering the availability of the phosphorylatable sites to the active sites on the protein kinases. Although this notion is supported by several lines of evidence, at present a role of the influence of polyamines on both the substrate and enzyme cannot be ruled out. Possible physiological relevance of the polyamine-stimulable protein kinase reactions observed in the in vitro experiments remains problematic in the absence of precise knowledge on the "effective" or free concentrations of intracellular polyamines.
Animals↗
Characteristics of polyamine stimulation of cyclic nucleotide-independent protein kinase reactions.
The extent of direct stimulation by spermine of reactions catalysed by nuclear N1 and N2 protein kinases purified from liver and prostate depends critically on the nature of the protein substrate. The chemically inert Co(NH3)36+ ion exerts effects on protein kinase reactions similar to those of spermidine or spermine. This enhancement of the phosphorylation of various protein substrates by polyamines or Co(NH3)63+ by purified nuclear protein kinase preparations was studied in relation to effects of temperature, pH and other factors. The results provide further support for our hypothesis [Ahmed, Wilson, Goueli & Williams-Ashman (1978) Biochem. J. 176, 739-750] that the enhancement of certain protein kinase reactions by polycations relates primarily to their interaction with the protein substrate, yielding more favourable conformations for phosphorylation by the protein kinase, rather than a direct effect on its catalytic activity.
Animals↗
Polyamine-stimulated phosphorylation of prostatic spermine-binding protein is mediated only by cyclic AMP-independent protein kinases.
Spermine-binding protein (a rat ventral prostatic protein with high affinity for spermine) was phosphorylated in situ through the action of intrinsic cellular protein kinase(s), suggesting it to be a phosphoprotein in vivo. The purified protein served as a substrate in a number of cyclic AMP-independent protein kinase reactions in vitro, but not for cyclic AMP-dependent, Ca2+ + calmodulin-dependent or Ca2+ + phospholipid-dependent protein kinases. Available data indicate that at least one of the cyclic AMP-independent protein kinases (cytosolic protein kinase C2) may be physiologically relevant in mediating the phosphorylation of this protein. The phosphorylation reaction was stimulated several-fold in the presence of spermine. Spermidine was somewhat less effective, whereas putrescine, cadaverine and 1,6-hexanediamine were minimally active. Phospho amino acid analysis of 32P-labelled spermine-binding protein indicated that phosphoserine was the only labelled phospho amino acid. Spermine-binding protein did not undergo autophosphorylation, or modify the stimulative effect of spermine on the phosphorylation of other substrates such as non-histone proteins. In situ the phosphorylation of spermine-binding protein in tissue from castrated rats was markedly diminished as compared with the normal. Since the phosphorylation of spermine-binding protein appears to be mediated by cyclic AMP-independent protein kinase(s) whose activity in the prostate is under androgenic control, it is suggested that androgen-dependent modulation of the protein kinase(s) exerts a regulatory control (via phosphorylation-dephosphorylation) on the spermine-binding activity and stability of this protein in vivo. Further, since this protein is a substrate for only the cyclic AMP-independent protein kinases, it could serve as a tool for the investigation of such kinases.
Amino Acids↗
Chromatin-associated protein kinases in human normal and benign hyperplastic prostate.
Nuclear phosphoproteins and protein kinases of human normal and benign hyperplastic prostate (BPH) were studied in an effort to delineate their properties and to identify any underlying differences therein. Chromatin-associated protein kinases active towards phosvitin, lysine-rich histone, and endogenous nonhistone proteins were characterized in human prostatic nuclei. The general properties of the human chromatin-associated prostatic protein kinases were similar to those of rat ventral prostate chromatin. Polyamines stimulated the phosphorylation of endogenous nonhistone proteins and phosvitin. Protein kinases active towards phosvitin and lysine-rich histones were unaltered in chromatin from BPH tissue as compared with the normal prostate. However, phosphorylation of chromatin-associated nonhistone proteins was markedly enhanced (average, 123%) in BPH tissue as compared with the normal tissue. The results indicate a change in the protein kinase reaction specifically involving chromatin-associated nonhistone proteins of BPH tissue as compared with normal human prostate.
Adenosine Triphosphate↗
Phosphorylation of prostatic nuclear matrix proteins is under androgenic control.
Nuclear matrix fraction was isolated from rat ventral prostatic nuclei previously incubated with [gamma-32P]ATP to label nuclear phosphoproteins with 32P. A significant portion of the radioactivity was recovered in the phosphoproteins intrinsic to the nuclear matrix fraction. At 12 h after androgen deprivation (i.e., when a significant portion of the nuclear androgen receptor was known to be depleted), the rate, but not the extent, of phosphorylation of nuclear proteins (predominantly nonhistone proteins) was markedly reduced. Nuclear matrix fraction isolated from such preparations demonstrated a profound reduction in the rate of incorporation of 32P into the matrix-associated proteins without any apparent change in the gel electrophoretic profile of these proteins. The results indicate that the cAMP-independent protein kinase activity which catalyzes the phosphorylation of nuclear matrix proteins is under androgenic control. This may be germane to nuclear matrix-associated initial events in androgen action.
Androgens↗
Phosphorylation of the androgen receptor by a nuclear cAMP-independent protein kinase.
The androgen receptor was purified from rat ventral prostate. The purified receptor migrated as a single band of mol. wt. 87000 on SDS-polyacrylamide gels, had a kd for R-1881 (17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one) binding as 6 nM, and sedimentation coefficient of 4.5 S. Phosphorylation of the purified receptor was studied by incubating it with [gamma-32P]ATP in the presence of several purified protein kinases including cAMP-dependent protein kinase, and four cAMP-independent protein kinases (which were active towards substrates such as phosvitin and casein). Phosphorylation of the 87000 mol. wt. androgen receptor protein occurred only in the presence of a nuclear cAMP-independent protein kinase (of the N2 type). No auto-phosphorylation of the receptor was detected. The results indicate that the androgen receptor is a phosphoprotein. Further, phosphorylation of the androgen receptor by only a specific nuclear cAMP-independent protein kinase may be important in determining the dynamics of its function.
Animals↗
Differential androgenic control of prostatic cytosolic cAMP-dependent and -independent protein kinases.
Whether or not various cytosolic protein kinases (and especially the type I cAMP-dependent protein kinase) of rat ventral prostate are specifically regulated with respect to total activity or specific activity by androgen has been investigated. Following androgen deprivation, the total activity per prostate of cAMP-dependent protein kinase (with histone as substrate) changed little at 24 h, declining by about 20% at 96 h. Under these conditions, its specific activity remained unaltered at 24 h, but was markedly enhanced at 96 h postorchiectomy. Type II cAMP-dependent protein kinase in rat ventral prostate cytosol was the only form of cAMP-dependent protein kinases present as determined by measurement of catalytic activity as well as [32P]-8-N3-cAMP binding to the regulatory subunits. There was no alteration in the distribution of the isoenzymes of cAMP-dependent protein kinases or the response of these kinase activities to cAMP owing to castration of animals. The prostatic cytosol also contains free regulatory subunit (with molecular weight similar to that of regulatory subunit R1) which coelutes with type II cAMP-dependent protein kinase. This finding was confirmed by using [32P]-8-N3-cAMP photoaffinity labeling of cAMP-binding proteins. With respect to cAMP-independent protein kinase (measured with dephosphophosvitin as substrate), a decline of 31% in its specific activity was observed in cytosol of prostates from rats castrated for a period of 24 h without significant further change at later periods following castration. However, there was a marked progressive reduction in total activity of this enzyme per prostate (loss of 72% at 96 h postorchiectomy). The increase in specific activity of cAMP-dependent, but not cAMP-independent, protein kinase in the face of decreasing total activity in the cytosol at later periods of castration (e.g., at 96 h) may reflect a slower loss of the former enzyme protein than the bulk of the cytosolic proteins. Administration of testosterone to castrated animals prevented these changes. These data do not indicate a specific regulation by steroid of the type I cAMP-dependent protein kinase in the prostate. Rather, the cAMP-independent protein kinase (with dephosphophosvitin as substrate) appears to be modulated by the androgenic status of the animal.
Androgens↗
Polyamine-like effects of cobalt (III) hexaammine on various cyclic nucleotide-independent protein phosphokinase reactions.
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Animals↗
Differential effects of polyamines on the phosphorylation of chromatin-associated proteins.
Studies are presented on the nature of chromatin-associated phosphoproteins whose phosphorylation is influenced by polyamines. After labelling with 32P, chromatin-associated proteins were separated into four fractions. Fraction I comprised neutral and basic non-histone phosphoproteins, including high-mobility-group non-histones; fraction II consisted mostly of histones; fraction III consisted of a class of (salt-soluble) acidic non-histone phosphoproteins; and fraction IV consisted of residual (salt-insoluble) acidic non-histone phosphoproteins. The average relative distribution of protein in the four fractions (I-IV) was about 1:4:2:1 for both liver and prostate. However, tissue-dependent differences were observed in the incorporation of 32P in various protein fractions. In the presence of polyamines (e.g. 1 mM-spermine or 2 mM-spermidine) maximal stimulation of phosphorylation was observed in non-histone proteins of fraction I (160-180%), followed by that in non-histone proteins of fraction III (80-110%). The phosphorylation of residual non-histone proteins in fraction IV, and the small extent of phosphorylation of histones in fraction II, remained unaltered in the presence of polyamines. Thus polyamines do not stimulate the phosphorylation of all non-histone proteins; their stimulative effect is most prominent in the phosphorylation of neutral and basic non-histone proteins and a class of salt-soluble acidic non-histone proteins. In accord with our hypothesis, these differential effects of polyamines on phosphorylation of endogenous non-histone proteins may relate to the conformation of these substrates rather than to endogenous kinases.
Animals↗