Muscarinic agonists and phorbol esters increase tyrosine phosphorylation of a 40-kilodalton protein in hippocampal slices.

Abstract

We have used the hippocampal slice preparation to investigate the regulation of protein tyrosine phosphorylation in brain. After pharmacological treatment ofintact slices, proteins were separated by electrophoresis, and levels of protein tyrosine phosphorylation were assessed by immunoblotting with specific anti-phosphotyrosine antibodies. Phorbol esters, activators of the serine-and threonine-phosphorylating enzyme protein kinase C, selectively increase tyrosine phosphorylation of a soluble protein with an apparent molecular mass of approximately 40 kilodaltons. Muscarinic agonists such as carbachol and oxotremorine M that strongly activate the inositol phospholipid system also increase tyrosine phosphorylation of this protein. Neurotransmitter activation of the inositol phospholipid system and protein kinase C appears to trigger a cascade leading to increased tyrosine phosphorylation.In recent years, a group of protein kinases has been identified that selectively phosphorylates proteins on tyrosine residues (1). These phosphotyrosine residues are much less abundant than phosphoserine or phosphothreonine residues, accounting for <1% of phosphoamino acid residues in many cell types. Despite the low levels of phosphotyrosine-containing proteins, tyrosine-specific protein kinases play a major role in regulating cell processes, since several oncogene products and growth factor receptors possess tyrosine-specific protein kinase activity critical for influencing cell growth and proliferation (1, 2).Tyrosine kinase activity is particularly abundant in adult brain (3, 4). Paradoxically, it is associated with neurons, a nonproliferating cell type (5-8), indicating involvement in aspects of neuronal function other than cell proliferation. Several synaptic vesicle proteins (7) and the nicotinic acetylcholine receptor (9) have been identified as substrates for tyrosine phosphorylation. These findings point to a role of tyrosine-specific kinases in synaptic transmission, yet little is known about the regulation of tyrosine phosphorylation in neuronal systems. In the present study, we have used the hippocampal slice preparation to examine this question. We have found that tyrosine phosphorylation of a 40-kilodalton (kDa) protein is increased by both phorbol esters and muscarinic receptor activation, indicating that tyrosine phosphorylation may mediate some responses to neurotransmitters and protein kinase C activation. MATERIALS AND METHODSHippocampal Slice Preparation. Rat hippocampal slices were prepared in a standard fashion (10) from adult male Sprague-Dawley rats (150-250 g). Hippocampi were dissected, and 400-,m-thick transverse slices were cut with a manual chopper. Slices were transferred to a chamber containing a humidified atmosphere of 5% C02/95% 02. Slices rested on filter paper covering a small dish of physiological saline (130 mM NaCl/5.0 mM KCI/24 mM NaHCO3/2.5 mM CaCl2/1.5 mM MgSO4/1.2 mM NaH2PO4/10 mM glucose). Slices were allowed to recover for at least 1 hr prior to transferring them to dishes with...

Angiotensin II stimulates protein-tyrosine phosphorylation in a calcium-dependent manner.

“…These include thrombin (22,30,46), collagen (50), and vasopressin (32) in platelets, fMetLeuPhe in neutrophils (39), muscarinic agonists in hippocampal slices (64), and phytohemagglutinin in Jurkat T cells (69). Disparate findings have been reported regarding the signaling pathways involved in this atypical tyrosine phosphorylation.…”

Activation of the genetically defined m1 muscarinic receptor potentiates N -methyl- d -aspartate (NMDA) receptor currents in hippocampal pyramidal cells

“…Recent studies reveal that the src family of tyrosine kinases can potentiate I NMDA and that this may be mediated by phosphorylation of tyrosine in the C-terminal domain of the NR2A subunit (51)(52)(53)(54). Interestingly, activation of muscarinic receptors can activate tyrosine kinases in hippocampal slices (60,61), suggesting that m1-induced activation of tyrosine kinase could mediate the m1-induced potentiation of I NMDA . However, future studies will be needed to rigorously test this hypothesis.…”

A critical period of protein kinase activity after tetanic stimulation is required for the induction of long-term potentiation.

“…By modulating spontaneous synaptic activity in neuronal cultures with extracellular Ca 2+ and tetrodotoxin (TTX), Murphy et al (1994) observed that constitutive MAPK activity increased slowly (10 min to maximal levels) with synaptic stimulation and decreased gradually (20 min to return to baseline) after the addition of TTX. MAPK must be phosphorylated on tyrosine and threonine residues to become constitutively active, which is mediated by a PKC-regulated activator kinase (Stratton et al 1989). In contrast, CaMKII activation was rapid (10 sec) and decayed to baseline within 3 min after TTX treatment.…”