Possible involvement of Fyn kinase in ethanol-stimulated Cas tyrosine phosphorylation in rat cerebellum and cerebral cortex

In the present study, we have investigated the effect of intraperitoneal injection of ethanol (3.5 g/kg) on tyrosine phosphorylation in rat brain. Immunoblot analysis using an antiphosphotyrosine antibody revealed that a 130-kDa protein band was detected in the brain extract in response to ethanol administration. This ethanol-stimulated tyrosine phosphorylation of the 130-kDa protein was found in the brain but not in the heart, liver or thymus. The 130-kDa phosphotyrosine-containing protein was identified by immunoprecipitation to be Cas, a crk-associated src substrate. This ethanol-stimulated tyrosine phosphorylation of Cas was observed most prominently in the cerebellum and the cerebral cortex. We further examined the possible involvement of Fyn kinase in ethanol-stimulated Cas tyrosine phosphorylation. Immunecomplex kinase assay showed that Fyn was activated in the cerebellum and cerebral cortex of ethanol-administered rats. Immunoprecipitation experiments also showed that Fyn was co-immunoprecipitated with an anti-Cas antibody in these regions from ethanol-administered rats. Furthermore, exogenous Fyn was shown to phosphorylate Cas from cerebellum and cerebral cortex in vitro. These findings indicate that ethanol stimulates tyrosine phosphorylation of Cas in rat cerebellum and cerebral cortex, and that Fyn may be involved in the process.     

Identification of p42 Mitogen-Activated Protein Kinase as a Tyrosine Kinase Substrate Activated by Maximal Electroconvulsive Shock in Hippocampus

Abstract: Recent studies have demonstrated that administration of an electroconvulsive shock produces a rapid and transient increase in tyrosyl phosphorylation of a ∼40-kDa protein in rat brain. Initial characterization of this protein's chromatographic properties indicated that it might be a member of a recently identified family of kinases, referred to as mitogen-activated protein (MAP) kinases, that are activated by tyrosyl phosphorylation. In the present study, we have used MAP kinase antisera to assess the identity of this protein. We have found that the ∼40-kDa phosphotyrosine-containing protein comigrates with p42 MAP kinase (p42^mapk) and not with two other 44-kDa MAP kinase family members detected by these antisera. Western blots of proteins immunoprecipitated with MAP kinase antibodies confirm that p42^mapk displays increased tyrosyl phosphorylation after an electroconvulsive stimulus. Chromatographic separation of hippocampal extracts indicates that MAP kinase activity elutes in parallel with p42^mapk. Accordingly, these studies identify p42^mapk as a tyrosyl kinase substrate that is activated by this stimulus and suggest that this form of MAP kinase may be selectively regulated by neuronal stimulation.     

Changes of protein tyrosine phosphorylation in third instar larval integument of the Mediterranean fruit fly, Ceratitis capitata

Developmental analysis of the tyrosine protein phosphorylation levels in larval integument and partial characterization of the endogenous protein tyrosine kinase activity (PTK) in Ceratitis capitata are described in this study. Larval integument contains high levels of PTK activity at the early stages of the third instar, which progressively declines to low levels in the white pupal stage. An integumental 90-kDa polypeptide was identified to have prominent endogenous PTK activity and follow a similar developmental pattern. The major integumental phosphotyrosine-containing polypeptides have apparent molecular weights of 30, 41, 44, 46, and 54 kDa, respectively. Polypeptides with molecular weights of 62 and 73 kDa were identified as Ser/Thr-containing phosphoproteins and were shown to exhibit high levels of phosphorylation at the middle stage of larval development. These differences are likely to be due to the higher activation state of the protein tyrosine kinase(s) at the early stages of larval development.     

Activation and Tyrosine Phosphorylation of 44-kDa Mitogen-Activated Protein Kinase (MAPK) Induced by Electroconvulsive Shock in Rat Hippocampus

Abstract: Electroconvulsive shock (ECS) has been reported to induce the phosphorylation and activation of 42-kDa, but not 44-kDa, mitogen-activated protein kinase (MAPK) in rat hippocampus. We studied the activation and tyrosine phosphorylation of MAPKs in rat brain after ECS. We observed the increase of the activities of both 42- and 44-kDa MAPKs in rat hippocampus after ECS. The activities reached peak at 2 min and returned to basal levels by 15 min after ECS. We also observed the increased phsophorylation on the tyrosine residue of 42-kDa MAPK in rat hippocampus after ECS, but not on that of 44-kDa MAPK. However, when we examined the immunoprecipitated 44-kDa MAPK, we could demonstrate that the tyrosine phosphorylation of 44-kDa MAPK at 2 min after ECS was markedly increased, in accordance with the increase of kinase activity. These results indicate that ECS induces the transient activation and tyrosine phosphorylation of 44-kDa MAPK, as well as 42-kDa MAPK, in rat hippocampus, although the amount of tyrosine phosphorylation is far less and the kinase activity is lower in 44-kDa MAPK than in 42-kDa MAPK.     

Phosphatase Resistance of ERK2 Brain Kinase PK40erk2

Abstract: We have previously shown that a brain protein kinase, termed PK40, catalyzes the multiple phosphorylation of the KSP-repeat site of neurofilaments (NFs) and also can transform τ proteins into the paired helical filament-like state as found in Alzheimer's disease (AD) brains. Protein sequence analysis suggests that PK40 is a form of the extracellular signal-regulated kinase ERK2. A subpopulation of ERK2 species in soluble brain fractions can be efficiently phosphorylated and activated in cell-free systems, simply by adding Mg²⁺-ATP. Two phosphoisoforms of PK40^erk2 are formed in this process, which have a reduced gel mobility, very much like the ERK2 form obtained in cell culture by stimulation with growth factors. One of these low-mobility forms cannot be inactivated with protein phosphatase 2A (PP2A) or with tyrosine phosphatases. The second form can be slowly inactivated by PP2A. In this case two Ser/Thr phosphates are removed at different rates during inactivation: One phosphate is very quickly removed to result in the formation of a high-mobility 39-kDa ERK2 species without consequence for activity; the other, slowly removed Ser/Thr phosphate controls the activity but has no effect on the gel mobility of ERK2. These results show that forms of ERK2 exist with properties different from the previously characterized ERK2 (p42^mapk) from stimulated cell cultures. The active ERK2 forms produced in the presence of Mg²⁺-ATP alone could provide an explanation for the existence of constitutive ERK2-like NF phosphorylation in vivo. Excessive formation of an ERK2 species resistant to inactivation by PP2A might be relevant to the persistent pathological τ hyperphosphorylation in AD.     

Tyrosine Phosphorylation of Microtubule-Associated Protein Kinase After Transient Ischemia in the Gerbil Brain

The tyrosine phosphorylation of microtubule-associated protein (MAP) kinase was examined in the gerbil brain after transient ischemia and reperfusion. Phosphorylation of MAP kinase was maximal within 1 min of reperfusion following 5 min of ischemia and returned to control levels as early as 5 min postischemia. The greatest increase in MAP kinase phosphorylation was detected in the hippocampus, with minor increases in other ischemic regions of the brain. Several tyrosine-phosphorylated proteins were detected in the gerbil hippocampus; however, the ischemia and reperfusion injury only increased tyrosine phosphorylation of MAP kinase. The increase in tyrosine phosphorylation was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker (+)-MK-801, whereas a non-NMDA receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione, was ineffective. Pretreatment of gerbils with calcium channel blockers also prevented the tyrosine phosphorylation of MAP kinase in the ischemic brain. Altogether, these results imply an involvement of glutamate receptors and calcium during the tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation was also prevented when ischemia and reperfusion were conducted under hypothermic conditions, which protect against neurodegenerative damage. These findings implicate a role for MAP kinase in neuronal damage resulting from ischemia and reperfusion.     

Protein Kinase FA/GSK-3 Phosphorylates on Ser235-Pro and Ser404-Pro that Are Abnormally Phosphorylated in Alzheimer's Disease Brain

Abstract— Previously, we identified protein kinase F_A/gly-cogen synthase kinase-3 (GSK-3) as a microtubule-associated protein kinase that can incorporate 4 mol of phosphates into 1 mol of protein and cause its electrophoretic mobility shift in sodium dodecyl sulfate gels, a unique property characteristic of paired helical filament-associated pathological (PHF-) in Alzheimer's disease brains. In this report, we identified TPPKS(p)PSAAK and SPVVSGDTS(p)PR as two phosphorylation site sequences phosphorylated by kinase F_A/GSK-3 in using peptide sequence analysis and sequential manual Edman degradation for radiosequencing. When mapping with human brain sequence, we further identified Ser²³⁵-Pro and Ser⁴⁰⁴-Pro as the two major phosphorylation sites according to the numbering of the longest isoform. Ser²³⁵ and Ser⁴⁰⁴ have been reported as two of the major abnormal phosphorylation sites in PHF-. Taken together, the results provide initial evidence that protein kinase F_A/GSK-3 may represent one of the Ser-Pro motif-directed kinases involved in the abnormal phosphorylation of pathological PHF- in Alzheimer's disease brain.     

Identification of an Activator of the Microtubule-Associated Protein 2 Kinases ERK1 and ERK2 in PC12 Cells Stimulated with Nerve Growth Factor or Bradykinin

Treatment of PC12 pheochromocytoma cells with nerve growth factor (NGF) or bradykinin leads to the activation of extracellular signal-regulated kinases ERK1 and ERK2, two isozymes of microtubule-associated protein 2 (MAP) kinase that are present in numerous cell lines and regulated by diverse extracellular signals. The activation of MAP kinase is associated with its phosphorylation on tyrosine and threonine residues, both of which are required for activity. In the present studies, we have identified a factor in extracts of PC12 cells treated with NGF or bradykinin, named MAP kinase activator, that, when reconstituted with inactive MAP kinase from untreated cells, dramatically increased MAP kinase activity. Activation of MAP kinase in vitro by this factor required MgATP and was associated with the phosphorylation of a 42- (ERK1) and 44-kDa (ERK2) polypeptide. Incorporation of 32P into ERK1 and ERK2 occurred primarily on tyrosine and threonine residues and was associated with a single tryptic peptide, which is identical to one whose phosphorylation is increased by treatment of intact PC12 cells with NGF. Thus, the MAP kinase activator identified in PC12 cells is likely to be a physiologically important intermediate in the signaling pathways activated by NGF and bradykinin. Moreover, stimulation of the activator by NGF and bradykinin suggests that tyrosine kinase receptors and guanine nucleotide-binding protein-coupled receptors are both capable of regulating these pathways.     

Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.     

A cyclic adenosine 3',5'-monophosphate-induced tyrosine phosphorylation of Syk protein tyrosine kinase in the flagella of boar spermatozoa

A cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein tyrosine phosphorylation is involved in the expression of fertilizing ability in mammalian spermatozoa. However, there are only limited data concerning the identification of protein tyrosine kinase (PTK) that is activated by the cAMP signaling. In this study, we have shown data supporting that boar sperm flagellum possesses a unique cAMP-protein kinase A (PKA) signaling cascade leading to phosphorylation of Syk PTK at the tyrosine residues of the activation loop. Ejaculated spermatozoa were washed and then incubated in a modified Krebs-Ringer HEPES medium (mKRH) containing polyvinyl alcohol (PVA) plus 0.1 mM cBiMPS (a cell-permeable cAMP analog), 0.25 mM sodium orthovanadate (Na3VO4) (a protein tyrosine phosphatase (PTP) inhibitor) or both at 38.5 degrees C for 180 min. Aliquots of the sperm suspensions were recovered before and after incubation and then used to detect sperm tyrosine-phosphorylated proteins by Western blotting and indirect immunofluorescence. In the Western blotting, the anti-phosphotyrosine monoclonal antibody (4G10) recognized several bands including 72-kDa protein in the protein extracts from spermatozoa that were incubated solely with cBiMPS. The tyrosine phosphorylation in these sperm proteins was dependent on cBiMPS and enhanced by the addition of Na3VO4. The 72-kDa tyrosine-phosphorylated protein was apparently reacted with the anti-phospho-Syk antibody (Tyr525/526). Indirect immunofluorescence revealed that the connecting and principal pieces of spermatozoa incubated with cBiMPS and Na3VO4 were stained with the anti-phospho-Syk antibody. However, the reactivity of the 72-kDa protein with the anti-phospho-Syk antibody was reduced by the addition of H-89 (a PKA inhibitor, 0.01-0.1 mM) to the sperm suspensions but not affected by the pretreatment of spermatozoa with BAPTA-AM (an intracellular Ca2+ chelator, 0.1 mM). Fractionation of phosphorylated proteins from the spermatozoa with a detergent Nonidet P-40 suggested that the 72-kDa tyrosine-phosphorylated protein might be a cytoskeletal component. Based on these findings, we have concluded that the cAMP-PKA signaling is linked to the Ca2+-independent tyrosine phosphorylation of Syk in the connecting and principal pieces of boar spermatozoa.     

Identification of Ca2+/calmodulin-dependent protein kinase and endogenous substrate of Fusarium oxysporum

Ca2+/calmodulin-dependent phosphorylation and cross-reactivity between anti-rat brain Ca2+/calmodulin-dependent protein kinase II (CaMK) antibody and partially purified CaMK from Fusarium oxysporum were detected in the component of high-molecular mass (M(r) greater than 100,000). In vitro, Ca2+/CaM-dependent phosphorylation of only a 16-kDa protein was detected. The 16-kDa protein was localized in the membrane fraction. Amino acid sequence of one of the peptides derived from partial hydrolysis of the 16-kDa protein had a high homology (65.5%) with the bovine transducin beta chain. It is assumed that the 16-kDa protein is an endogenous substrate of F. oxysporum CaMK.     

Protein-tyrosine phosphorylations induced by concanavalin A and N-formyl-methionyl-leucyl-phenylalanine in human neutrophils.

The ability of the lectin concanavalin A (ConA) and N-formyl-methionyl-leucyl-phenylalanine (fMLF) to induce protein-tyrosine phosphorylation in human neutrophils was examined by immunoblot analysis. ConA caused an increase in tyrosine phosphorylation of protein bands with apparent molecular masses of 120, 80, 76, 66 and 40 kDa; on the other hand, fMLF caused an increase in those of only 80-kDa and 40-kDa proteins. These protein-tyrosine phosphorylations were time- and dose-dependent. The tyrosine phosphorylation of 40-kDa protein induced by fMLF was suppressed but that by ConA was not suppressed by pertussis toxin pretreatment. At the same time, pertussis toxin pretreatment also inhibited lysozyme release and aggregation of neutrophils induced by fMLF but did not inhibit those responses induced by ConA. These results suggest that the tyrosine phosphorylation of 40-kDa protein may be involved in a part of neutrophil activation and be regulated via pleiotropic signal transduction pathways. In addition, immunoblot analysis employing antibodies against microtubule-associated protein 2 (MAP2) kinase suggested that this tyrosine-phosphorylated 40-kDa protein might be the MAP2 kinase.     

Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation.

Despite extensive study of sperm motility, little is known of the mechanism of mammalian sperm hyperactivation. Here we describe a novel method for preparation of rodent sperm flagella and use it to show a correlation between tyrosine phosphorylation of flagellar proteins and hyperactivation of hamster sperm. When hyperactivation was produced by a 3.5-h incubation in a medium supporting capacitation, four major tyrosine-phosphorylated peptides of 90-, 80-, 62-, and 48-kDa mass were detected in flagellar extracts. Incubation with calyculin A, an inhibitor of protein phosphatases 1 and 2A, produced hyperactivation within 40 min but only a single 80-kDa phosphotyrosine-containing flagellar component. Conversely, incubation with inhibitors of either protein kinase A (H8) or protein tyrosine kinase (tyrphostin 47) prevented both hyperactivation and the production of tyrosine-phosphorylated flagellar peptides. These results indicate a strong correlation of hyperactivation with the tyrosine phosphorylation of sperm flagellar peptides, and they strongly implicate an 80-kDa component as a major mediator of the mechanism that produces hyperactivated motility of hamster sperm.     

K-252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH-SY5Y Cells

Abstract: The protein kinase inhibitor K-252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K-252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH-SY5Y. K-252a induced neurite outgrowth in a dose-dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125- and 140-kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down-regulation of protein kinase C by long-term treatment with phorbol ester did not prevent K-252a-induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF-109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K-252a was also observed in LA-N-5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C-independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K-252a-induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.     

Cyclic AMP-Dependent Protein Kinase Decreases γ-Aminobutyric AcidA Receptor-Mediated 36Q1− Uptake by Brain Microsacs

The effect of cyclic AMP (cAMP)-dependent protein phosphorylation on gamma-aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol-stimulated 36Cl- uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP-dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol-stimulated 36Cl- uptake by approximately 25%. In parallel experiments, PKA and [gamma-32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitate the entire GABAA receptor complex, under nondenaturing conditions, using an anti-alpha 1-subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66-kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti-alpha 1 subunit antibody. Phosphopeptide mapping of the 66-kDa polypeptide demonstrated a 14-kDa fragment similar to that obtained with the purified, PKA-phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain GABAA receptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.     

Region-Specific Targets of p42/p44MAPK Signaling in Rat Brain

Abstract: In vitro studies indicate that p42/p44^MAPK phosphorylate both nuclear and cytoplasmic proteins. However, the functional targets of p42/p44^MAPK activation in vivo remain unclear. To address this question, we localized activated p42/p44^MAPK in hippocampus and cortex and determined their signaling effects after electroconvulsive shock treatment (ECT) in rats. Phosphorylated p42/p44^MAPK content increased in the cytoplasm of hippocampal neurons in response to ECT. Consistent with this cytoplasmic localization, inhibition of ECT-induced p42/p44^MAPK activation by the extracellular signal-regulated kinase kinase inhibitor PD098059 blocked phosphorylation of the cytoplasmic protein microtubule-associated protein 2c (MAP2c), but failed to inhibit the induction of the nuclear protein c-Fos in response to ECT. In contrast to hippocampal neurons, cortical neurons exhibited an increase in amount of phosphorylated p42/p44^MAPK in both the nucleus and cytoplasm after ECT. Accordingly, PD098059 blocked the induction of Fos-like immunoreactivity in the nuclei of cortical neurons as well as MAP2c phosphorylation in the cytoplasm. Our data indicate that both nuclear and cytoplasmic substrates can be activated by p42/p44^MAPK in vivo. However, the functional targets of p42/p44^MAPK signaling depend on the precise location of p42/p44^MAPK within different subcellular compartments of brain regions. These results indicate unique functional pathways of p42/p44^MAPK-mediated signal transduction within different brain regions in vivo.     

Time-Resolved Signaling Pathways of Nerve Growth Factor Diverge Downstream of the p140trk Receptor Activation Between Chick Sympathetic and Dorsal Root Ganglion Sensory Neurons

Abstract: We have recently shown that the small GTP binding protein p21 ras is essential for nerve growth factor (NGF)-mediated survival of peripheral embryonic chick dorsal root ganglia (DRG) sensory but not sympathetic neurons. To investigate at which level of the signaling cascade the pathways diverge, we have studied the time-resolved pattern of NGF-stimulated tyrosine phosphorylation of proteins within 4 h after addition of the neurotrophin. In both chick sympathetic neurons [embryonic day (E) 12] and DRG sensory neurons (E9) NGF induces within 1 min the autophosphorylation of the receptor tyrosine kinase p140trk. However, the pattern of substrate protein tyrosine phosphorylation downstream of p140trk is distinctly different in both neuronal subtypes. In sympathetic neurons, we observe within 1 min the tyrosine phosphorylation of a new substrate protein, p105, reaching maximal levels at 3 min. Tyrosine phosphorylation of p105 remains elevated for up to 4 h. Subsequent to p105, NGF induces the tyrosine phosphorylation of p42, a protein belonging to the family of mitogen-activated protein (MAP) kinases. This stimulation is transient, reaching maximal levels at 10 min and returning to very low levels already after 2 h. In DRG sensory neurons, tyrosine phosphorylation of p105 is weak and very short lived, disappearing already after treatment with NGF for 10 min. In contrast, activation of MAP kinase p42 in DRG sensory neurons is more stable than in sympathetic neurons. All NGF-stimulated tyrosine phosphorylation events were inhibited by preincubation of neurons with the tropomyosin-related kinase (trk) inhibitor K252a. We suggest the working hypothesis that persistent tyrosine phosphorylation of p105 may play a role in the p21ras-independent NGF survival pathway of chick sympathetic neurons.     

The tyrosine-phosphorylated hepatocyte growth factor/scatter factor receptor associates with phosphatidylinositol 3-kinase.

The receptor for hepatocyte growth factor, also known as scatter factor (HGF/SF), has recently been identified as the 190-kDa heterodimeric tyrosine kinase encoded by the MET proto-oncogene (p190MET). The signaling pathway(s) triggered by HGF/SF are unknown. In A549 cells, a lung epithelial cell line, nanomolar concentrations of HGF/SF induced tyrosine phosphorylation of the p190MET receptor. The autophosphorylated receptor coprecipitated with phosphatidylinositol 3-kinase (PI 3-kinase) activity. In GTL16 cells, a cell line derived from a gastric carcinoma, the p190MET receptor, overexpressed and constitutively phosphorylated on tyrosine, coprecipitated with PI 3-kinase activity and with the 85-kDa PI 3-kinase subunit. In these cells activation of protein kinase C or the increase of intracellular [Ca2+] inhibits tyrosine phosphorylation of the p190MET receptor as well as the association with both PI 3-kinase activity and the 85-kDa subunit of the enzyme. In an in vitro assay, tyrosine phosphorylation of the immobilized p190MET receptor was required for binding of PI 3-kinase from cell lysates. These data strongly suggest that the signaling pathway activated by the HGF/SF receptor includes generation of D-3-phosphorylated inositol phospholipids.     

Splicing Pattern of Gsα mRNA in Human and Rat Brain

The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.     

GSK-3 is activated by the tyrosine kinase Pyk2 during LPA1-mediated neurite retraction

Glycogen synthase kinase-3 (GSK-3) is a multifunctional serine/threonine kinase that is usually inactivated by serine phosphorylation in response to extracellular cues. However, GSK-3 can also be activated by tyrosine phosphorylation, but little is known about the upstream signaling events and tyrosine kinase(s) involved. Here we describe a G protein signaling pathway leading to GSK-3 activation during lysophosphatidic acid (LPA)-induced neurite retraction. Using neuronal cells expressing the LPA(1) receptor, we show that LPA(1) mediates tyrosine phosphorylation and activation of GSK-3 with subsequent phosphorylation of the microtubule-associated protein tau via the G(i)-linked PIP(2) hydrolysis-Ca(2+) mobilization pathway. LPA concomitantly activates the Ca(2+)-dependent tyrosine kinase Pyk2, which is detected in a complex with GSK-3beta. Inactivation or knockdown of Pyk2 inhibits LPA-induced (but not basal) tyrosine phosphorylation of GSK-3 and partially inhibits LPA-induced neurite retraction, similar to what is observed following GSK-3 inhibition. Thus, Pyk2 mediates LPA(1)-induced activation of GSK-3 and subsequent phosphorylation of microtubule-associated proteins. Pyk2-mediated GSK-3 activation is initiated by PIP(2) hydrolysis and may serve to destabilize microtubules during actomyosin-driven neurite retraction.