Madeleine Toutant

Regulation of a neuronal form of focal adhesion kinase by anandamide

Anandamide is an endogenous ligand for central cannabinoid receptors and is released after neuronal depolarization. Anandamide increased protein tyrosine phosphorylation in rat hippocampal slices and neurons in culture. The action of anandamide resulted from the inhibition of adenylyl cyclase and cyclic adenosine 3,5-monophosphate-dependent protein kinase. One of the proteins phosphorylated in response to anandamide was an isoform of pp125-focal adhesion kinase (FAK+) expressed preferentially in neurons. Focal adhesion kinaseis a tyrosine kinase involved in the interactions between the integrins and actin-based cytoskeleton. Thus, anandamide may exert neurotrophic effects and play a role in synaptic plasticity.

FAK and PYK2/CAKβ in the nervous system : a link between neuronal activity, plasticity and survival?

A major aim of neurobiology today is to improve understanding of the signaling pathways that couple rapid events, such as the action potential and neurotransmitter release, to long-lasting changes in synaptic strength and increased neuronal survival. These adaptations involve interactions of neurons with other cells and with the extracellular matrix. They use, in part, the same molecular machinery that controls adhesion, motility or survival in non-neuronal cells. This machinery includes two homologous non-receptor tyrosine kinases, FAK and PYK2/CAKbeta, and the associated SRC-family tyrosine kinases. Specific brain isoforms of FAK with distinct properties are regulated by neurotransmitters, whereas PYK2/CAKbeta is highly sensitive to depolarization. The multiplicity of the pathways that can be activated by these tyrosine kinases indicates their importance in signal transduction in the adult brain.

Sphingosine-1-phosphate induces proliferation of astrocytes: regulation by intracellular signalling cascades

Sphingosine‐1‐phosphate (S1P) is a potent lysophospholipid mediator mostly released by activated platelets. It is involved in several functions in peripheral tissues, but its effects in the central nervous system are poorly documented. Therefore, we have examined the effects of S1P on the proliferation of striatal astrocytes from the mouse embryo. These cells have been found to express mRNAs for the S1P receptors, Edg‐1 and Edg‐3. S1P stimulated thymidine incorporation and induced activation of extracellular signal‐regulated kinases (Erks). Both effects were prevented by U0126, an Erk kinase inhibitor. The S1P‐evoked activation of Erk1 was totally blocked in astrocytes pretreated with a combination of either phorbol ester (24 h) and LY294002, or phorbol ester (24 h) and pertussis toxin (PTX). Each individual treatment only partially inhibited Erk1 activation. This suggests that several separate mechanisms mediate this process, one involving protein kinase C and another involving Gi/Go proteins and phosphatidylinositol 3‐kinase. In contrast, the stimulatory effect of S1P on astrocyte proliferation was totally blocked by either PTX or LY294002, but not by a downregulation of protein kinase C. S1P dramatically inhibited the evoked production of cyclic AMP, a response that was impaired by PTX. Finally, S1P stimulated the production of inositol phosphates and increased intracellular calcium by mobilization from thapsigargin‐sensitive stores. These latter effects were mainly insensitive to PTX. Probably, Gi/Go protein activation and phosphoinositide hydrolysis are early events that regulate the activation of Erks by S1P. Altogether, these observations show that astrocytes are targets for S1P. Their proliferation in response to S1P could have physiopathological consequences at sites of brain lesions and alterations of the blood–brain barrier.

Differential Regulation of Proline-rich Tyrosine Kinase 2/Cell Adhesion Kinase β (PYK2/CAKβ) and pp125FAK by Glutamate and Depolarization in Rat Hippocampus

The mechanisms by which stimuli that raise cytosolic free Ca2+ concentrations in neurons can increase protein tyrosine phosphorylation are not known. Using rat hippocampal slices and cortical synaptosomes, we have examined the regulation of two highly related cytoplasmic tyrosine kinases, pp125 focal adhesion kinase (pp125FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase β (PYK2/CAKβ). Membrane depolarization increased tyrosine phosphorylation of PYK2/CAKβ and pp125FAK. These effects were blocked by EGTA or by protein kinase C inhibitors (RO31-8220; GF109203X) and mimicked by ionomycin or phorbol 12-myristate 13-acetate, in the case of pp125FAK, or their combination in the case of PYK2/CAKβ. Glutamate and specific agonists of ionotropic (α-amino-3-hydroxy-5-methyl-4-isoxazole propionate and N-methyl-D-aspartate) or metabotropic (trans-1-aminocyclopentane-1,3,-dicarboxylate) glutamate receptors stimulated the phosphorylation of pp125FAK, but not of PYK2/CAKβ. Glutamate effects were prevented by GF109203X. Thus, in hippocampal slices, tyrosine phosphorylation of pp125FAK and PYK2/CAKβ are regulated differentially by pathways involving Ca2+ and protein kinase C. pp125FAK and PYK2/CAKβ may provide specific links between neuronal activity, increases in cytosolic Ca2+ and protein tyrosine phosphorylation, which may be important for neuronal survival, and synaptic plasticity.

Presence of three pertussis toxin substrates and Goα immunoreactivity in both plasma and granule membranes of chromaffin cells

GTP‐binding proteins have been proposed to be involved in some secretory processes. Bordetella pertussis toxin is known to catalyze ADP‐ribosylation of several GTP‐binding proteins. In this paper, the subcellular localization of B. pertussis toxin substrates has been explored in chromaffin cells of bovine adrenal medulla. With appropriate gel electrophoresis conditions, three ADP‐ribosylated substrates of 39, 40 and 41 kDa were detectable in both plasma and granule membranes. The more intense labelling occurred on the 40 kDa component, while the 41 kDa species exhibited electrophoretic mobility similar to that of Giα. Significant immunoreactivity with anti‐Goα antibodies was detected at the level of the 39 kDa faster component. The association of G‐proteins with granule and plasma membranes suggests the involvement of these proteins in the exocytotic process or in its regulation.

Alternative Splicing Controls the Mechanisms of FAK Autophosphorylation

ABSTRACT Focal adhesion kinase (FAK) is activated following integrin engagement or stimulation of transmembrane receptors. Autophosphorylation of FAK on Tyr-397 is a critical event, allowing binding of Src family kinases and activation of signal transduction pathways. Tissue-specific alternative splicing generates several isoforms of FAK with different autophosphorylation rates. Despite its importance, the mechanisms of FAK autophosphorylation and the basis for differences between isoforms are not known. We addressed these questions using isoforms of FAK expressed in brain. Autophosphorylation of FAK+, which is identical to that of “standard” FAK, was intermolecular in transfected cells, although it did not involve the formation of stable multimeric complexes. Coumermycin-induced dimerization of gyrase B-FAK+ chimeras triggered autophosphorylation of Tyr-397. This was independent of cell adhesion but required the C terminus of the protein. In contrast, the elevated autophosphorylation of FAK+6,7, the major neuronal splice isoform, was not accounted for by transphosphorylation. Specifically designed immune precipitate kinase assays confirmed that autophosphorylation of FAK+ was intermolecular, whereas autophosphorylation of FAK+6,7 or FAK+7 was predominantly intramolecular and insensitive to the inhibitory effects of the N-terminal domain. Our results clarify the mechanisms of FAK activation and show how alternative splicing can dramatically alter the mechanism of autophosphorylation of a protein kinase.

PIAS1-mediated sumoylation of focal adhesion kinase activates its autophosphorylation

Focal adhesion kinase (FAK) is a protein tyrosine kinase enriched in focal adhesions, which plays a critical role in integrin-dependent cell motility and survival. The crucial step in its activation is autophosphorylation on Tyr-397, which promotes the recruitment of several enzymes including Src family kinases and the activation of multiple signaling pathways. We found in a yeast two-hybrid screen that the N-terminal domain of FAK interacted with protein inhibitor of activated STAT1 (PIAS1). This interaction was confirmed and shown to be direct using in vitro assays. PIAS1 was co-immunoprecipitated with FAK from transfected cells and brain extracts. PIAS1 has recently been recognized as a small ubiquitin-like modifier (SUMO) ligase. In the presence of PIAS1 and SUMO-1, FAK was sumoylated in intact cells, whereas PYK2, a closely related enzyme, was not. Sumoylation occurred on Lys-152, a residue conserved in FAK during evolution. Sumoylated FAK, like PIAS1, was recovered predominantly from the nuclear fraction. Sumoylation did not require the catalytic activity or autophosphorylation of FAK. In contrast, sumoylation increased dramatically the ability of FAK to autophosphorylate in intact cells and in immune precipitate kinase assays. Endogenous FAK was sumoylated in the presence of PIAS1 and SUMO-1 independently of cell adhesion, and autophosphorylation of sumoylated FAK was persistently increased in suspended cells. These observations show that sumoylation controls the activity of a protein kinase and suggest that FAK may play a novel role in signaling between the plasma membrane and the nucleus.

DIFFERENTIAL REGULATION OF FAK+ AND PYK2/CAKBETA , TWO RELATED TYROSINE KINASES, IN RAT HIPPOCAMPAL SLICES : EFFECTS OF LPA, CARBACHOL, DEPOLARIZATION AND HYPEROSMOLARITY

FAK+, an isoform of focal adhesion kinase preferentially expressed in brain and PYK2/Cakβ (proline‐rich tyrosine kinase 2/cell adhesion kinaseβ) are two related cytoplasmic tyrosine kinases. They are candidates for coupling electrical activity and stimulation of neurotransmitter receptors to short and long‐term changes in synaptic properties, cytoskeletal organization and gene expression in neurons. As the same set of stimuli appear capable of stimulating FAK and/or PYK2 in non‐neuronal cells and in cell lines with neuronal characteristics, we investigated the selectivity of regulation of these two kinases in mature nervous tissue. Using rat hippocampal slices, we compared the regulation of FAK+ and PYK2 by stimuli known to be active on one or the other of these two kinases in other cell types: lysophosphatidic acid (LPA), carbachol, depolarization, and hyperosmolarity. Phosphorylation of FAK+ was markedly increased by carbachol and LPA. Carbachol effects occurred via activation of M1 muscarinic receptors and nicotinic receptors. The effects of carbachol and LPA were prevented by protein kinase C inhibitors, whereas 8‐Br‐cAMP attenuated the effects of carbachol but not of LPA. Tyrosine phosphorylation of PYK2 but not of FAK+ was very strongly enhanced by depolarization and hyperosmolarity. This study and our previous results show that FAK+ and PYK2 are regulated differentially in hippocampal slices: FAK+ is phosphorylated on tyrosine in response to stimulation of G protein‐coupled receptors, whereas PYK2 is mainly sensitive to depolarization and hyperosmolarity. Thus, FAK+ and PYK2 may provide specific and separate links between activation of neurotransmitters receptors, depolarization and tyrosine phosphorylation in mature hippocampus.

Alternatively Spliced Focal Adhesion Kinase in Rat Brain with Increased Autophosphorylation Activity

pp125 focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase transducing signals initiated by integrin engagement and G protein-coupled receptors, is highly expressed in brain. FAK from brain had a higher molecular weight and an increased autophosphorylation activity, than from other tissues. In addition to a 9-base insertion in the 3′-coding region, which defines FAK+, rat striatal FAK mRNAs contained several additional short exons, coding for peptides of 28, 6, and 7 residues, respectively (termed boxes 28, 6, and 7), surrounding the autophosphorylated Tyr-397. In transfected COS 7 cells, the presence of boxes 6 and 7 conferred an increased overall tyrosine phosphorylation, a higher phosphorylation of Tyr-397 assessed with a phosphorylation state-specific antibody, and a more active autophosphorylation in immune precipitates. The presence of box 28 did not alter further these parameters. Two-dimensional phosphopeptide maps of hippocampal FAK were identical to those of FAK+6,7. The presence of the various exons did not alter the interaction of FAK with c-Src, n-Src, or Fyn. Thus, several splice isoforms of FAK are preferentially expressed in rat brain, some of which have an increased autophosphorylation activity, suggesting that FAK may have specific properties in neurons.

Pleiotropic effects of lysophosphatidic acid on striatal astrocytes.

Lysophosphatidic acid (LPA) is a potent lipid mediator that is likely involved in diverse functions in the brain. Several recent studies have suggested that astrocytes are important target cells for LPA. In the present study, we have identified the signal transduction pathways activated following LPA stimulation in mouse striatal astrocytes in primary culture. In cells prelabeled with myo‐[3H]inositol, LPA stimulated the formation of [3H]inositol phosphates (EC50 = 0.7 μM). This effect was reproduced neither by other lysophospholipids nor by phosphatidic acid. Astrocyte pretreatment with pertussis toxin partially abolished this LPA response indicating the involvement of a Gi/Go protein. In [3H]adenine‐prelabeled cells, LPA strongly inhibited the formation of [3H]cyclic AMP induced by forskolin (EC50 = 0.3 μM) and by isoproterenol and PACAP‐38. These inhibitory effects were strongly reduced by pertussis toxin treatment. Although with a lesser potency (EC50 = 5 μM), LPA also stimulated the release of [3H]arachidonic acid from [3H]arachidonic acid‐prelabeled astrocytes. This latter effect was totally inhibited by mepacrine, did not involve a pertussis toxin‐sensitive G protein, and was highly dependent on external calcium. LPA also stimulated the activity of both extracellular signal‐regulated kinases (Erk) Erk1 and Erk2 by a mechanism involving a Gi/Go protein. Surprisingly, in contrast to that observed in fibroblasts, LPA was totally ineffective in stimulating DNA synthesis. These results provide additional evidence in favor of an important physiological role of LPA in the astrocytic functions. GLIA 28:25–33, 1999.