Type IIβ Regulatory Subunit of cAMP-Dependent Protein Kinase: Purification Strategies to Optimize Crystallization

To elucidate the structural basis for important differences between types I and II regulatory subunit isoforms (RI and RII) of adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase, the full-length RIIβ isoform and five RIIβ deletion mutants were constructed, expressed, purified, and screened for crystallization. Only one of these six proteins yielded diffraction quality crystals. Crystals were grown of the RIIβ deletion mutant (Δ1–111) monomer potentially in complex with two cAMP molecules. X-ray diffraction quality data were obtained only after significant modification to existing purification procedures. Modifications required a Sepharose, not agarose, support for cAMP affinity chromatography followed by rapid, quantitative removal of free cAMP by size-exclusion chromatography under reducing conditions. Data to 2.4 Å resolution were collected at 29°C using synchrotron radiation on a single crystal measuring 0.2 × 0.3 × 1.2 mm³. Data were 99% complete. The hexagonal crystal belonged to space group P6₍₁₎ or P6₍₅₎ with unit cell dimensions a = b = 161.62 Å and c = 39.66 Å.     

Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta.

The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.     

Crystal structures of RIalpha subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase complexed with (Rp)-adenosine 3',5'-cyclic monophosphothioate and (Sp)-adenosine 3',5'-cyclic monophosphothioate, the phosphothioate analogues of cAMP

Cyclic adenosine 5'-monophosphate (cAMP) is an ancient signaling molecule, and in vertebrates, a primary target for cAMP is cAMP-dependent protein kinase (PKA). (R(p))-adenosine 3',5'-cyclic monophosphothioate ((R(p))-cAMPS) and its analogues are the only known competitive inhibitors and antagonists for cAMP activation of PKA, while (S(p))-adenosine 3',5'-cyclic monophosphothioate ((S(p))-cAMPS) functions as an agonist. The crystal structures of a Delta(1-91) deletion mutant of the RIalpha regulatory subunit of PKA bound to (R(p))-cAMPS and (S(p))-cAMPS were determined at 2.4 and 2.3 A resolution, respectively. While the structures are similar to each other and to the crystal structure of RIalpha bound to cAMP, differences in the dynamical properties of the protein when (R(p))-cAMPS is bound are apparent. The structures highlight the critical importance of the exocyclic oxygen's interaction with the invariant arginine in the phosphate binding cassette (PBC) and the importance of this interaction for the dynamical properties of the interactions that radiate out from the PBC. The conformations of the phosphate binding cassettes containing two invariant arginine residues (Arg209 on domain A, and Arg333 on domain B) are somewhat different due to the sulfur interacting with this arginine. Furthermore, the B-site ligand together with the entire domain B show significant differences in their overall dynamic properties in the crystal structure of Delta(1-91) RIalpha complexed with (R(p))-cAMPS phosphothioate analogue ((R(p))-RIalpha) compared to the cAMP- and (S(p))-cAMPS-bound type I and II regulatory subunits, based on the temperature factors. In all structures, two structural solvent molecules exist within the A-site ligand binding pocket; both mediate water-bridged interactions between the ligand and the protein. No structured waters are in the B-site pocket. Owing to the higher resolution data, the N-terminal segment (109-117) of the RIalpha subunit can also be traced. This strand forms an intermolecular antiparallel beta-sheet with the same strand in an adjacent molecule and implies that the RIalpha subunit can form a weak homodimer even in the absence of its dimerization domain.     

Biochemical and immunological characterization of the flagellar-associated regulatory subunit of a type II cyclic adenosine 5'-monophosphate-dependent protein kinase

We have shown previously that the regulatory subunit (RII) of a type II cyclic AMP (cAMP)-dependent protein kinase is tightly associated with mammalian sperm flagella (J. A. Horowitz et al. (1984) J. Biol. Chem. 259, 832-838; J. A. Horowitz et al. (1988) J. Biol. Chem. 263, 2098-2104). In the present study the flagellar RII was compared to other well-characterized RIIs using biochemical and immunological methods. Flagellar polypeptides were screened by immunoblot analysis with monoclonal antibodies directed against the RII alpha and RII beta isoforms. An RII beta monoclonal antibody failed to cross-react with any flagellar polypeptide. In contrast, mAB 622, an RII alpha/RII beta monoclonal antibody, cross-reacted with a 57,000 Da polypeptide. However, another RII alpha/RII beta monoclonal antibody interacted weakly with the flagellar RII, suggesting that the epitope for this antibody is modified in flagellar RII. Partial peptide mapping of 8-azido-[32P]cAMP-labeled RIIs revealed that although heart and testis generated similar fragmentation patterns, there were differences in the maps from flagellar RII. Two-dimensional sodium dodecyl sulfate-gel electrophoresis of 8-azido-[32P]cAMP-labeled RII from rat flagella and bovine heart showed that the former possessed a considerably more acidic isoelectric point. Partial proteolysis of the flagellar RII by either endogenous or exogenous proteases resulted in the cleavage of RII to a 40,000 Mr fragment. Complete release of this fragment from the flagellum was achieved if proteolysis was performed in the presence of thiol reducing agents. In their absence, approximately 50% of the fragment remained bound to the flagellum. The soluble proteolytic fragment was shown to be monomeric by native high-resolution gel-permeation chromatography and contained a functional cAMP-binding site(s).     

Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum.

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N3[32P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RI alpha (Mr 47,000) subunits, a prominent type II holoenzyme containing RII beta (Mr 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RII beta and RII alpha (Mr 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RI alpha protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RII beta holoenzyme in very close proximity to free RI alpha and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RI alpha.     

Manganese(II) complex of 6,7-dicycanodipyridoquinoxaline with antitumor activities: synthesis,crystal structure and binding with DNA

A new Mn(II) complex with the planar ligand 6,7-dicycanodipyrido[2,2-D:2',3'-f]quinoxaline (L) [MnL(NO(3))(H(2)O)(3)]NO(3).CH(3)OH (1) has been synthesized and characterized by elemental analysis, IR, TG-DTA and molar conductance. Its crystal structure was determined by X-ray diffraction, crystal data: yellow, triclinic, space group P1;, Z=2, a=7.3743(8) A, b=11.2487(15) A, c=14.1655(15) A, alpha=79.412(2) degrees, beta=83.208(2) degrees, gamma=80.466(2) degrees. The Mn atom was hexa-coordinated to form a distorted octahedral geometry by two nitrogen atoms of L and four oxygen atoms of three H(2)O and NO(3)(-) in the complex. The binding mode of the complex with calf thymus DNA has also been investigated with spectrophotometric methods, viscosity and thermal denaturation measurements. The experimental results indicate that the complex intercalated into DNA base pairs via the ligand L. The intrinsic binding constant K(b) values for 1 (5.00 x 10(5) M(-1)) and L (1.65 x 10(5) M(-1)) were determined by absorption titration and calculated with the model of McGhee and Von Hippel. Biological tests against four different cell lines (HL-60, KB, Hela and BGC-823) in vitro showed that the complex had significant antitumor properties since the 50% inhibition concentrations (IC(50)) of the complex were within a microM range similar to those of antitumor drug 5-fluorouracil.     

Molecular cloning, cDNA structure and deduced amino acid sequence for the hormone-induced regulatory subunit (RII beta) of cAMP-dependent protein kinase from rat ovarian granulosa cells

The regulatory subunit of cAMP-dependent protein kinase designated RII beta (RII51) has previously been shown to be the product of a separate gene. This was accomplished by the molecular cloning of a partial cDNA clone estimated to lack 30-45 nucleotides of the 5' end of the coding region. We hereby report the isolation of a cDNA clone for RII beta from rat granulosa cells, extending 43 nucleotides further 5' compared with the previously published cDNA sequence, and from which the entire amino acid sequence (415 residues) of the rat RII beta protein can be deduced. A cAMP regulated mRNA of 3.2 kilobases (kb) for RII beta was detected by the isolated cDNA in rat Sertoli cells.     

L-Leucinamide hydrogensquarate: spectroscopic and structural elucidation

The hydrogensquarate [LeuNH(2)] (HSq) of L-leucinamide has been synthesized and its structure has been determined by single crystal X-ray diffraction. A three dimensional network is formed by hydrogen bonds with participation of the O=C-NH(2) function, the hydrogensquarate ion and the N(+)H(3) group [NH(2)...O=C((Sq)) (2.840 and 2.749 A), ((Sq))OH...O=C(NH(2)) (2.618 A), NH(3) (+)...O=C((Sq)) (3.246, 2.804 and 2.823 A)], respectively. A theoretical approximation of the electronic structure was carried out by means of ab initio UMP2 and MP2 level of theory at the 6-311++G** basis set. The IR-spectroscopic assignment in the solid-phase was obtained by linear-polarized IR-spectroscopy of oriented samples as colloid suspensions in a nematic host and application of the reducing-difference procedure for the interpretation of polarized IR-spectra.     

Identification of the MAP2- and P75-binding domain in the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase. Cloning and expression of the cDNA for bovine brain RII beta

cDNA clones coding for the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a bovine brain cDNA expression library in lambda gt11. The cDNA codes for a protein of 418 amino acids which is 98% homologous to the rat and human RII beta proteins. A series of expression vectors coding for truncated RII beta proteins were constructed in pATH plasmids and fusion proteins were expressed in Escherichia coli. Polyclonal and monoclonal antibodies made against purified bovine brain RII were immunoreactive with the fusion proteins on Western blots. The expressed RII beta-fusion proteins were used in overlay assays to identify the region in RII beta which binds to microtubule-associated protein 2 (MAP2) and to the 75,000-dalton calmodulin-binding protein (P75) (Sarkar, D., Erlichman, J., and Rubin, C.S. (1984) J. Biol. Chem. 259, 9844-9846) in bovine brain. Fusion protein containing amino acids 1-50 of the RII beta NH2 terminus (RII beta(1-50)] bound to both MAP2 and P75 immobilized on nitrocellulose filters. A pATH11-directed fusion protein containing the 31 amino acid RII-binding site of the human MAP2 protein (MAP2(31)) (Rubino, H.M., Dammerman, M., Shafit-Zagardo, B., and Erlichman, J. (1989) Neuron 3, 631-638) also bound RII beta-fusion proteins containing RII beta amino acids 1-50. Three fusion proteins, RII beta(1-25), RII beta(25-96), and RII beta(1-265,25-96 deleted) did not bind to MAP2(31) nor P75. The results showed that the binding domain for MAP2 and P75 was located within the NH2-terminal 50 amino acids of RII beta. Preincubation of bovine heart protein kinase II alpha and RII beta(1-50) with MAP2(31) prevented their binding to both P75 and MAP2(31) that were immobilized on nitrocellulose, suggesting that the binding sites for MAP2 and P75 are located near each other or that the same site on RII was binding to both proteins.     

The structure at 2 A resolution of Phycocyanin from Gracilaria chilensis and the energy transfer network in a PC-PC complex

Phycocyanin is a phycobiliprotein involved in light harvesting and conduction of light to the reaction centers in cyanobacteria and red algae. The structure of C-phycocyanin from Gracilaria chilensis was solved by X-ray crystallography at 2.0 A resolution in space group P2(1). An interaction model between two PC heterohexamers was built, followed by molecular dynamic refinement. The best model showed an inter-hexamer rotation of 23 degrees . The coordinates of a PC heterohexamer (alphabeta)(6) and of the PC-PC complex were used to perform energy transfer calculations between chromophores pairs using the fluorescence resonance energy transfer approach (FRET). Two main intra PC ((I)beta(3)(82)-->(I)alpha(1)(84)-->(I)alpha(5)(84)-->(I)beta(6)(82) and (I)beta(3)(153)-->(I)beta(5)(153)) and two main inter PC ((I)beta(6)(82)-->(II)beta(3)(82) and (I)beta(5)(153)-->(II)beta(3)(153)) pathways were proposed based on the values of the energy transfer constants calculated for all the chromophore pairs in the hexamer and in the complex.     

Crystallization and preliminary X-ray analysis of a fungal endoglucanase I.

An endoglucanase I (EG1) from a fungal source (Humicola insolens) has been crystallized in a number of forms suitable for X-ray diffraction analysis. Four crystal forms have been grown from various precipitants using vapour phase diffusion methods in hanging drops. Three of these crystal forms diffract to beyond 2.5 A resolution. Two forms, obtained from ammonium sulphate at pH 5.4, or 8.0, grow as tetragonal bipyramids in space group P4(1)22 or P4(3)22, with approximate cell dimensions a = b = 102 A, c = 282 A. The other crystal forms were grown from polyethylene glycol 8000 at pH 8.0. One grows as monoclinic plates, space group P2(1), with cell dimensions a = 66.9 A, b = 75.2 A, c = 86.9 A and beta = 102.9 degrees and the other as long hexagonal rods in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 119 A, c = 83 A.     

The regulatory subunit of the type II cAMP-dependent protein kinase in rabbit ovaries is the RII beta isoform.

Based on RII autophosphorylation, photoaffinity labeling with 8-N3[32P]cAMP, and Western blot analysis we have identified the RII isoform found in rabbit corpora lutea as RII beta. The RII beta subunit found in rabbit corpora lutea differs from the RII beta found in rat follicles and corpora lutea in that it migrates at Mr 52,500 on SDS-PAGE and shifts to Mr 53,000 when phosphorylated.     

Structure-activity studies of alpha-melanotropin fragments on cAMP production in striatal slices

In this work, we characterized the active site in the alpha-melanotropin hormone (alpha-MSH) sequence responsible for the enhancement of cAMP production in incubated striatal slices by using different alpha-MSH fragments. We also analyzed the effects of the co-incubation of the SCH23390, a dopaminergic D(1) antagonist, with the MSH fragments, to study the involvement of the D(1) receptor on this induction. A rise was observed in the levels of cAMP after addition of the 6 microM fragments MSH((1-10)), and 0.6 and 6 microM MSH((5-13)); however, the values were lower than those induced by 6 microM alpha-MSH. On the contrary, the addition of MSH((9-13)), MSH((7-11)), or MSH((6-9)) did not affect the cAMP content. The presence of 10 microM SCH23390 blocked the effect of the fragments on cAMP production. We conclude that the biologic activity of alpha-MSH, as observed through the levels of cAMP, declines when the length of its polypeptide chain is shortened, and that the presence of glutamic acid in the molecule, as well as the core sequence, are of importance for fragments' activity.     

Glucose uptake during centrally induced stress is insulin independent and enhanced by adrenergic blockade.

Glucose utilization increases markedly in the normal dog during stress induced by the intracerebroventricular (ICV) injection of carbachol. To determine the extent to which insulin, glucagon, and selective (alpha/beta)-adrenergic activation mediate the increment in glucose metabolic clearance rate (MCR) and glucose production (R(a)), we used five groups of normal mongrel dogs: 1) pancreatic clamp (PC; n = 7) with peripheral somatostatin (0.8 microg x kg(-1) x min(-1)) and intraportal replacement of insulin (1,482 +/- 84 pmol x kg(-1) x min(-1)) and glucagon (0.65 ng x kg(-1) x min(-1)) infusions; 2) PC plus combined alpha (phentolamine)- and beta (propranolol)-blockade (7 and 5 microg x kg(-1) x min(-1), respectively; alpha+beta; n = 5); 3) PC plus alpha-blockade (alpha; n = 6); 4) PC plus beta-blockade (beta; n = 5); and 5) a carbachol control group without PC (Con; n = 10). During ICV carbachol stress (0-120 min), catecholamines, ACTH, and cortisol increased in all groups. Baseline insulin and glucagon levels were maintained in all groups except Con, where glucagon rose 33%, and alpha, where insulin increased slightly but significantly. Stress increased (P < 0.05) plasma glucose in Con, PC, and alpha but decreased it in beta and alpha+beta. The MCR increment was greater (P < 0.05) in beta and alpha+beta than in Con, PC, and alpha. R(a) increased (P < 0.05) in all groups but was attenuated in alpha+beta. Stress-induced lipolysis was abolished in beta (P < 0.05). The marked rise in lactate in Con, PC, and alpha was abolished in alpha+beta and beta. We conclude that the stress-induced increase in MCR is largely independent of changes in insulin, markedly augmented by beta-blockade, and related, at least in part, to inhibition of lipolysis and glycogenolysis, and that R(a) is augmented by glucagon and alpha- and beta-catecholamine effects.     

Molecular characterization of bovine brain P75, a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta

In mammalian brain, physiological signals carried by cAMP seem to be targeted to intraneuronal sites by the association of cAMP-dependent protein kinase II beta with anchoring proteins that bind the regulatory subunit (RII beta) of the enzyme. Previously, an RII beta-binding domain was characterized in a large (Mr approximately 150,000) candidate anchor protein, rat brain P150 (Bregman, D. B., Bhattacharyya, N., and Rubin, C. S. (1989) J. Biol. Chem. 264, 4648-4656). RII beta-binding proteins with Mr values of 65,000-80,000 were detected in the brains of other species. Since little was known about the structural features of these lower Mr proteins, we undertook the characterization of bovine brain P75 as a prototype. A cDNA encoding 258 amino acid residues at the C terminus of P75 was cloned by probing a lambda gt11 expression library with 32P-RII beta. The cDNA insert was ligated into the pET-3b expression plasmid, and large amounts of the partial P75 polypeptide (designated P47) were produced in Escherichia coli. A purification scheme that yielded 9 mg of soluble P47 from a 1-liter bacterial culture was devised. Antibodies directed against the P47 polypeptide revealed that P75 is expressed almost exclusively in brain. The sequence of 117 amino acid residues at the C terminus of P75 contains the RII beta-binding site and is 80% identical to the corresponding region of P150. In contrast, a lower level of identity (36%) between P75 and P150 at a more N-terminal region indicates that the two RII beta-binding proteins are related, but distinct proteins. P75 is not homologous to microtubule-associated protein 2, an RII alpha-selective binding protein, or any other previously studied proteins. C-terminal truncation analysis disclosed that the final 26 residues in P75 are essential for binding RII beta.     

Hormonal regulation of the synthesis and mRNA content of the regulatory subunit of cyclic AMP-dependent protein kinase type II in cultured rat ovarian granulosa cells

These studies were undertaken to determine the molecular events by which estradiol and follicle-stimulating hormone (FSH) stimulate in ovarian granulosa cells the increase in the content of one of the regulatory subunits of cAMP-dependent protein kinase type II, RII51 (Mr = 51,000), and its electrophoretic variants RII51.5 (Mr = 51,500) and RII52 (Mr = 52,000). To analyze the de novo synthesis of RII51/52, granulosa cells were cultured (10(6) cells/ml) for 0, 12, 24, or 48 h with estradiol (10 nM) +/- FSH (12.5, 25, and 50 ng/ml), 8-bromo-cAMP (0.25-3 mM), or forskolin (0.5-100 microM) and then pulse-labeled with [35S]methionine (300 microCi/ml; 4 h). Labeled RII51, present either in urea extracts of total cellular protein or after partial purification from a soluble cell extract by cAMP-Sepharose chromatography, was quantitated by autoradiography of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and by excision of the silver-stained spots of the RII51 variants from the gels and counting. Synthesis of RII51 and its electrophoretic variants was low in cells cultured with estradiol alone for 48 h, whereas it was increased 4-5-fold in cells cultured with estradiol and FSH. Changes in the synthesis of actin were minor throughout the culture period regardless of hormone treatment. Pulse-chase experiments using [35S]methionine provided evidence that the isoelectric variants RII51.5 and RII52 may be derived from RII51 by post-translation modification, such as phosphorylation. Labelling with [32P]orthophosphate showed that RII52 contained more radioactivity than RII51.5 and RII51. Northern and filter hybridization assays demonstrated a 6-10-fold dose- and time-dependent increase in the amount of RII51 mRNA in granulosa cells exposed to estradiol and FSH or estradiol and forskolin compared to those cultured with estradiol alone. In vitro translation of poly(A)+ mRNA of granulosa cells from estradiol- and FSH-treated hypophysectomized rats also demonstrated an increase in the content of translatable RII51 mRNA. These studies indicate that in cultured rat granulosa cells the synthesis of RII51 and the content of its mRNA are selectively increased by estradiol and cAMP in a time- and dose-dependent manner. Based on these observations, RII51 appears to be a useful marker to determine the molecular (genomic?) sites of estradiol and FSH action in differentiating rat granulosa cells.     

Regulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity and regulatory subunit RII beta content by basic fibroblast growth factor (bFGF) during granulosa cell differentiation: possible implication of protein kinase C in bFGF action.

We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation.