Naji Sahyoun

Nuclear and axonal localization of Ca2+/calmodulin-dependent protein kinase type Gr in rat cerebellar cortex.

The granule cell-enriched Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr) is a recently discovered neuron-specific enzyme. The kinase avidly phosphorylates synapsin I and contains a polyglutamate sequence, which suggests an association with chromatin as well. A possible role in synapsin I phosphorylation and in nuclear Ca2+ signaling was supported by immunochemical and ultrastructural examination of CaM kinase-Gr distribution. CaM kinase-Gr immunoreactivity was present in the molecular and granule cell layers of the rat cerebellum. This pattern corresponded to the occurrence of the enzyme in the granule cell axons and nuclei, respectively. Immunoblots confirmed these findings. Thus, CaM kinase-Gr may mediate and coordinate Ca2(+)-signaling within different subcellular compartments.

Protein kinase C: Rapid enzyme purification and substrate-dependence of the diacylglycerol effect

Protein kinase C has been purified by a rapid method resulting in a high-yield, stable enzyme preparation. The catalytic and regulatory properties of this enzyme preparation were characterized employing histone H1 and HMG8, a proteolytic fragment of H1. The enzyme had a lower Km for HMG8, and was stimulated more effectively by diacylglycerol and phorbol esters in the presence of this substrate. Furthermore, these activators markedly increased the Km for HMG8 but not for H1. Protein kinase C and cyclic AMP-dependent protein kinase phosphorylate serine residues which are located in different, single tryptic peptides from HMG8.

Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release.

Postsynaptic densities contain a subtype of protein kinase C

Protein kinase C or an isoenzyme thereof appears to be a significant component of postsynaptic densities (PSDs) from rat brain. This cytoskeletal organelle binds 4 beta-phorbol 12,13-dibutyrate (PDBu) with a Bmax of about 20 pmol/mg protein and an apparent Kd of 3.3 nM. Ca2+ and phosphatidyl serine (PS) stimulated the endogenous phosphorylation of a subset of PSD polypeptides with Mr values between 16,000 and 22,000. Finally, a monospecific protein kinase C antibody reacted with a Mr 70,000 PSD polypeptide which migrated on SDS-PAGE slightly ahead of the Mr 77,000 purified enzyme. These data suggest that protein kinase C or a similar enzyme can be integrated into a cytoskeletal system and may play an important role in postsynaptic function.

Early postnatal development of calmodulin-dependent protein kinase II in rat brain

The postnatal levels of the alpha- (Mr 50,000) and beta- (Mr 58,000/60,000) subunits of type II calmodulin-dependent protein kinase were investigated in cytosolic, cytoskeletal and nuclear subfractions of rat brain. Substantial amounts of the beta-subunit were present neonatally, especially in the cytoplasm; both subunits increased markedly during the subsequent three weeks with alpha-preponderance in the cortex and midbrain. The development of the alpha-subunit was attenuated in the cerebellum and brain stem, resulting in a relative excess of the beta-subunit. Primary neuronal cultures from fetal brain initially displayed low enzyme content, followed by an increase in the levels of the beta- but not alpha-subunit. Regulation of the cellular content of type II calmodulin-dependent protein kinase may be relevant to neuronal differentiation.

Protein kinase C binding to isolated nuclei and its activation by a Ca2+phospholipid-independent mechanism

The direct interaction of protein kinase C with the nucleus was examined utilizing endogenous protein phosphorylation and [3H]PDBu binding to detect the enzyme. Rat brain nuclei were relatively rich in phorbol ester receptors whereas liver nuclei contained less than 10% of their brain counterpart. Purified protein kinase C from rat brain could bind to purified rat liver nuclei at 4 degrees C or at 24 degrees C reaching apparent equilibrium by 20 min. The binding was linearly dependent on protein kinase C concentration and required free Ca2+ with an EC50 of 0.5 microM. Chelation of Ca2+ with EGTA resulted in rapid loss of phorbol ester receptors from nuclei. Differential extraction experiments with Triton X-100 and NaCl suggested that about 50% of the acquired phorbol ester receptors were bound to chromatin and 25% were associated with the nuclear matrix. Protein Kinase C bound to nuclei was also able to phosphorylate several endogenous nuclear substrates in a Ca2+/phospholipid-independent reaction. These data suggest that protein kinase C can associate with nuclear components leading to the phosphorylation of nuclear substrates.

Cytoskeletal association of the cholera toxin receptor in rat erythrocytes

Summary Cholera toxin receptors are associated with the cytoskeletal residue of Triton X-100 extracts of rat erythrocytes. The solubilized cytoskeletal toxin-receptor complex appears to be a large macromolecular entity whose formation is facilitated by increased receptor occupancy. These findings suggest a role for GM 1 -gangliosides in transmembrane signalling and in the mechanism of action of cholera toxin.

Evidence for cytoskeletal associations of the adenylate cyclase system obtained by differential extraction of rat erythrocyte ghosts

Abstract Subpopulations of adenylate cyclase and its GTP-binding regulatory component (N-protein) are associated with Triton-insoluble cytoskeletons of rat erythrocytes. The cytoskeletal association of adenylate cyclase appears to be a function of its state of activation. The mechanism of hormonal activation of this enzyme may involve cytoskeletal components.

Molecular resolution and reconstitution of the GPP(NH) and NAF sensitive adenylate cyclase system

Abstract When a special detergent-extraction procedure is applied to rat brain particulate fractions, the latters adenylate cyclase activity becomes virtually unresponsive to NaF or Gpp (NH)p (guanylyl-5′-imidodiphosphate) despite the fact that under these conditions the enzyme does not appear to be removed (i.e., solubilized) from the membranes. Addition of exogenous fractions of detergent-solubilized membranes or of water-soluble samples of homogenates, obtained from various tissues, restores the stimulation of the enzyme by both Gpp(NH)p and NaF. These findings indicate that the stimulation caused by these agents is mediated by one or more regulatory component(s), and that these are molecular components physically distinct from the enzyme itself. The regulatory component(s) appear to be proteinaceous in nature and sensitive to SH-reactive reagents. The properties of the reconstituted system resemble those of the original particulate adenylate cyclase. This system may serve as a convenient tool for the study of the molecular properties of adenylate cyclase and of the basis of its regulatory control.

Involvement of fodrin-binding proteins in the structure of the neuronal postsynaptic density and regulation by phosphorylation.

Novel polypeptides with Mr values about 140,000 bind fodrin and spectrin and are enriched in the postsynaptic density (PSD) compared to other tissues or subcellular fractions. 125I-fodrin binding to these polypeptides is competed for by unlabeled spectrin. These polypeptides are distinct from ankyrin and its proteolytic fragments and from band 4.1 which also bind fodrin. Phosphorylation of PSDs by the endogenous calmodulin-dependent protein kinase markedly reduces 125I-fodrin binding to the transblotted preparation. Such an event may play a regulatory role in governing protein-protein interactions among elements of the PSD.