Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones

• Published in: Journal of neurochemistry 2004-03, Vol.88 (5), p.1127-1139
• Main Authors: Crossthwaite, Andrew J., Valli, Haseeb, Williams, Robert J.
• Format: Article
• Language: English
• Subjects: Akt/protein kinase B; Animals; Biochemistry and metabolism; Biological and medical sciences; Calcium - pharmacology; Cells, Cultured; Central nervous system; Corpus Striatum - cytology; Cyclic AMP Response Element-Binding Protein - metabolism; cyclic AMP response element‐binding protein; c‐Jun N‐terminal kinase; Enzyme Inhibitors - pharmacology; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Fundamental and applied biological sciences. Psychology; Glutamic Acid - metabolism; Glutamic Acid - pharmacology; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinases - metabolism; Neurons - cytology; Neurons - drug effects; Neurons - metabolism; NMDA; Phosphatidylinositol 3-Kinases - antagonists & inhibitors; Phosphatidylinositol 3-Kinases - metabolism; phosphatidylinositol 3‐kinase; Phosphorylation - drug effects; Protein Structure, Tertiary - physiology; Protein-Serine-Threonine Kinases; Protein-Tyrosine Kinases - metabolism; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-akt; Receptors, N-Methyl-D-Aspartate - metabolism; Signal Transduction - drug effects; src-Family Kinases - antagonists & inhibitors; src-Family Kinases - metabolism; tyrosine kinase; Vertebrates: nervous system and sense organs
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2004.02257.x

Glutamate receptor activation of mitogen‐activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+‐influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal‐regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3‐kinase (PI 3‐kinase)‐dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor‐induced phosphorylation of ERK1/2 and Akt/PKB in a concentration‐dependent manner, and reduced cAMP response element‐binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase‐mediated phosphorylation and PI 3‐kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3‐kinase regulatory subunit p85. This revealed a Src‐dependent phosphorylation of a focal adhesion kinase (FAK)–p85 complex on glutamate stimulation. Demonstrating that PI3‐kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3‐kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+‐dependent phosphorylation of c‐Jun N‐terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor‐dependent JNK1/2 phosphorylation, suggesting that Src family kinase‐dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3‐kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.