Viable mouse thymocytes as a model system for studying the onset of hormone-induced cellular refractoriness.

Mouse thymocytes are characterized as a model cellular system for studying the onset of hormone-induced cellular refractoriness (desensitization). This system has the following combination of useful features. (a) The cells can be isolated without the use of digestive enzymes, avoiding possible damage to surface receptors or to other exposed membranal constituents. (b) They can be kept viable for several hours, a period during which both stimulation and desensitization get well under way. (c) They can be stimulated by a variety of hormones which function via cAMP (beta-agonists, prostaglandin E1 and specific thymic humoral factors). (d) Their desensitization is receptor-specific. (e) They can be readily ruptured under mild conditions so as to allow a physiologically relevant biochemical analysis of hormonal stimulation and desensitization. (f) The hormonal response of these cells can be monitored simultaneously by the activation of adenylate cyclase, by the intracellular level of cAMP, and by the activation of cAMP-dependent protein kinase (which functions as a metabolic sensor for cAMP). In this cellular system, desensitization does not involve processes such as the efflux of cAMP, the activation of cAMP-phosphodiesterase or the synthesis of a protein mediator. On the other hand, desensitization can be accounted for by a hormone-triggered inactivation of the adenylate cyclase system. The immediate desensitization of thymocytes is reversible and occurs without apparent loss of functional receptors. Continuous presence of hormone is shown to be required not only for triggering the chain of events which leads to the readily reversible desensitization, but also for the process which transfers the cells to the subsequent, 'locked' desensitized state.

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation.

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

Vitronectin.

Vitronectin is a multifunctional glycoprotein present in blood and in the extracellular matrix. It binds glycosaminoglycans, collagen, plasminogen and the urokinase-receptor, and also stabilizes the inhibitory conformation of plasminogen activation inhibitor-1. By its localization in the extracellular matrix and its binding to plasminogen activation inhibitor-1, vitronectin can potentially regulate the proteolytic degradation of this matrix. In addition, vitronectin binds to complement, to heparin and to thrombin-antithrombin III complexes, implicating its participation in the immune response and in the regulation of clot formation. The biological functions of vitronectin can be modulated by proteolytic enzymes, and by exo- and ecto-protein kinases present in blood. Vitronectin contains an RGD sequence, through which it binds to the integrin receptor alpha v beta 3, and is involved in the cell attachment, spreading and migration. Antibodies against alpha v beta 3 or synthetic peptides containing an RGD sequence are now being tested as therapeutic agents in the treatment of human cancers, bone diseases (e.g. osteoporosis) and in pathological disorders which involve angiogenesis.

Evidence showing that the two-chain form of vitronectin is produced in the liver by a selective furin cleavage.

The adhesive protein vitronectin (75 kDa) occurs in human blood fluid in a one-chain (Vn75) or a two-chain form (Vn65-10), and is produced by a specific cleavage (at Arg379-Ala380), by a proteinase not identified hitherto. These two forms were shown to be functionally different and therefore, this cleavage may have a regulatory significance in vivo. Here, we report the use of a tailored one-chain recombinant Vn, a specific protein kinase A phosphorylation at Ser378, and sequence analysis to show: (1) that none of the proteinases originating from blood, previously thought to be the endogenous proteinase (plasmin, thrombin, tPA, and uPA), is indeed the in vivo convertase; and (2) that furin, a serine endoproteinase residing in the secretory pathway of hepatocytes, where Vn is synthesized, specifically cleaves Vn at the endogenous cleavage site. Consequently, we propose that the Vn75 to Vn65-10 conversion takes place in the liver (not in blood) and is carried out by furin.

The negative charge of Glu-127 in protein kinase A and its biorecognition.

A set of mutants of protein kinase A (PKA) in which Gln-127 was replaced by Gln, Asp, Asn, and Arg was prepared. Their Km and Vmax values show that the negative charge of Glu-127 (not merely its hydrogen bonding capacity) is indispensable for the kinase activity, since Glu-127/Gln is inactive, in spite of the fact that it can form hydrogen bonds and is very similar in bulkiness and conformation to wt-PKA. Glu-127 is involved in the biorecognition of PKA, interacting ionically with the positively charged guanido group of Arg P-3 (a major recognition element in the consensus sequence of PKA). In support of this conclusion, it is shown that a regression of the Glu-127 carboxylate by 1.54 A (as in Glu-127/Asp) results in an active kinase with a similar thermal stability and susceptibility to conformation-dependent proteolysis, a similar Vmax, an identical Km for ATP, but a > 20-fold higher Km for kemptide. The two inactive mutants of PKA, Glu-127/Gln and Glu-127/Asn, are potentially useful for studying protein-protein interactions of PKA, e.g. for monitoring enzymatically the displacement of active PKA from its complexes.

Disposition of the carboxy-terminus tail of rabbit lactase-phlorizin hydrolase elucidated by phosphorylation with protein kinase A in vitro and in tissue culture.

The intracellular disposition of the carboxy-terminus tail of rabbit lactase-phlorizin hydrolase (LPH) is demonstrated, using a specific phosphorylation of Ser1916 by protein kinase A (PKA). This phosphorylation is shown to occur not only in vitro (with pure LPH and pure catalytic subunit of PKA), but also in an organ culture of the small intestine. Cholera toxin, which is known to act in vivo on the membranes of the small intestine, with severe clinical consequences, and to elevate the intracellular cyclic AMP of enterocytes, is shown to enhance significantly the phosphorylation of LPH in intact cells grown as an organ culture. These findings establish the cytosolic orientation of the carboxy-terminus tail of LPH in situ, and raise the possibility that the tail itself and its phosphorylation by PKA may have a physiological or physiopathological significance.

Plasmin cleavage of vitronectin. Identification of the site and consequent attenuation in binding plasminogen activator inhibitor-1.

Plasmin is shown to specifically cleave vitronectin at the Arg361-Ser362 bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two-chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Ser378 with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type-1 inhibitor of plasminogen activator (PAI-1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAI-1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.

Anti-head and anti-tail antibodies against distinct epitopes in the catalytic subunit of protein kinase A. Use in the study of the kinase splitting membranal proteinase KSMP.

Protein kinases share a considerable sequence homology in their catalytic core (residues 40-300 in PKA). Each core is flanked by "head" and "tail" segments at its amino- and carboxy-termini, which are different in the various kinases. These end segments may play an important role in creating the preferential affinity of each kinase for its physiological substrates or regulatory ligands. Here we describe three anti-peptide antibodies (alpha P-1, alpha P-2, and alpha P-3) that specifically recognize the head and tail segments of the catalytic subunit (C) of PKA. (i) alpha P-1 (against 6A-K23) react with C when denatured but not when in its native structure; (ii) alpha P-2 (against 319K-I335), bind to the site in C cleaved by the kinase splitting membranal proteinase (KSMP) and inhibit this cleavage of C; (iii) alpha P-3 (against 338S-F350) react with C but not with the KSMP cleavage product C', useful for detecting a KSMP-like activity in different tissues and subcellular loci. The combined use of the antibodies described here provides a strict definition of C, and thus a high degree of fidelity in its biorecognition.

The phosphorylation of the two-chain form of vitronectin by protein kinase A is heparin dependent.

In circulating blood, vitronectin occurs in two forms: a single-chain (75 kDa) and an endogenously clipped two-chain form (65 kDa and 10 kDa) held together by a disulfide bridge. The 75 kDa form was previously shown to be phosphorylated at Ser378 by protein kinase A, released by physiologically stimulated platelets. By contrast, at pH 7.5 the two-chain form is not phosphorylated at all. Heparin or heparan sulfate are shown here to modulate the conformation of clipped vitronectin at physiological pH, exposing Ser378 and allowing its stoichiometric phosphorylation by the kinase. At this pH the two-chain form of vitronectin in plasma exhibits a higher affinity for heparin, and behaves as a flexible molecule, which can conformationally respond to heparin and heparan sulfate, effectors involved in vitronectin function.

A cAMP-triggered release of a hormone-like peptide.

Preparations of the catalytic subunit of cAMP-dependent protein kinase from rabbit skeletal muscle, which appear to be homogeneous by SDS-polyacrylamide gel electrophoresis, were often found to contain a hormone-like factor (HLF) which causes an immediate rise, then a decline of intracellular cAMP in a B-lymphoma cell line. Active HLF is released when the fractions that contain it in an inactive form are incubated with cAMP prior to chromatography, or passed through an immobilized cAMP column. HLF seems to be a peptide: it loses its cell-stimulating capability after proteolysis and has an apparent molecular mass of 2.2-2.5 kDa.

Differential expression of the protein kinase A regulatory subunit (RIalpha) in pancreatic endocrine cells.

A PCR-based subtractive cloning procedure was used to identify genes expressed at higher levels in the pancreatic beta cell line betaTC1, as compared to the pancreatic alpha cell line alphaTC1. One of the clones isolated by this procedure corresponded to the regulatory subunit (RIalpha) of protein kinase A (PKA). Using antibodies directed against RIalpha, we now demonstrate both by immunoblot and immunofluorescence that RIalpha protein is present at higher levels in cultured beta cells as compared to alpha cells. In vitro PKA assays revealed high basal PKA activity in alphaTC1 extracts, which changed little on addition of exogenous cAMP. On the other hand, extracts from beta cells showed very low basal activity of PKA, which was elevated upon addition of cAMP. A similar trend was observed in vivo using transfected luciferase constructs bearing multiple copies of a CRE element: in alphaTC1 cells, no induction by forskolin was observed, whereas in betaTC1 cells, forskolin produced a 9-fold increase in activity. Therefore, the results indicate that RIalpha of PKA is selectively expressed in pancreatic beta cells as compared to alpha cells: this selective expression is associated with major differences in the properties of the PKA signal transduction pathway. Differential expression of the regulatory subunit may play a role in determining the patterns of gene expression and signal transduction characteristic of alpha and beta cells.

Synthetic peptides derived from the sequence around the plasmin cleavage site in vitronectin. Use in mapping the PAI-1 binding site.

A series of 8 peptides derived from the amino acid sequence accommodating the plasmin cleavage site in vitronectin were synthesized and used to map its binding site for the type I plasminogen activator inhibitor (PAI-1). This mapping assigned the inhibitor binding site to the K348-R370 region with high affinity recognition elements within the K348-R357 sequence. These results account for our previous finding that cleavage of the R361-S362 bond by plasmin significantly reduces the affinity between PAI-1 and vitronectin, since it splits the PAI-1 binding site in two. Furthermore, in the case of the two-chain form of vitronectin, this cleavage detaches the S362-R379 peptide which provides some of the affinity elements for the binding of PAI-1.

A periodicity in the response of SJL/J thymocytes to isoproterenol. Simulation by cell lines.

Thymocytes from SJL/J mice exhibit a periodicity in their response to hormonal stimulation with isoproterenol. This periodicity (5-9 days) is expressed in large changes in the intensity of the response (peak levels of intracellular cAMP which vary approximately 6-fold), and in the response pattern, i.e., in the occurrence or non-occurrence of an immediate hormone-induced desensitization. In contrast, C57BL/6 thymocytes have a homogeneous response pattern (in all cases there was an immediate desensitization). Their response does change with the same periodicity, but these changes are restricted to the intensity of response (the peak of cAMP levels varies only approximately 2-fold). Using T, B and pre-B shown that these periodic changes may be due to fluctuations in T cell subpopulations in the thymus and possibly also the infiltration of B cells into it. These observations provide a useful system for studying at the cellular and molecular level the known correlation between endocrine disorders and neoplasia in mice. On the other hand, the set of cell lines with such different response patterns to a single hormone may be used for the identification and isolation of additional cellular constituents involved in the cellular response to hormones and its immediate desensitization.