Site-specific phosphorylation of tyrosine hydroxylase after KCl depolarization and nerve growth factor treatment of PC12 cells

The phosphorylation and activation of tyrosine hydroxylase was examined in PC12 cells following depolarization with KCl or treatment with nerve growth factor. Both treatments activate tyrosine hydroxylase (TH) and increase enzyme phosphorylation. Site-specific analysis of the tryptic phosphopeptides of TH isolated from [32P]phosphate-labeled PC12 cells demonstrated that the major phosphorylated peptide (termed "H25") did not contain any of the previously reported phosphorylation sites. Phosphoamino acid analysis of this peptide demonstrated that the phosphorylated residue was a serine. Synthetic tryptic peptides containing putative phosphorylation sites were prepared, and subjected to high performance liquid chromatography analysis and isoelectric focusing. The tryptic phosphopeptide containing serine 31 comigrated with the H25 peptide during both of these analytical techniques. The tryptic phosphopeptide produced by the phosphorylation of tyrosine hydroxylase by the recently discovered proline-directed protein kinase and the phosphorylated synthetic phosphopeptide TH2-12 are clearly separated from H25 by this analysis. We conclude that serine 31 is phosphorylated during KCl depolarization and nerve growth factor treatment of PC12 cells and that this phosphorylation is responsible for the activation of tyrosine hydroxylase. Since this site is not located in a sequence selective for any of the "classical" protein kinases, we suggest that a novel protein kinase may be responsible for the phosphorylation of this site. Since serine 31 has a proline residue on the carboxyl-terminal side, the possibility that this kinase may be related to the recently reported proline-directed protein kinase is discussed. Other sites that are also phosphorylated on TH during KCl depolarization include serine 19, which is known to be phosphorylated by calmodulin-dependent protein kinase II. A schematic model for the regulation of tyrosine hydroxylase activity by phosphorylation of the NH2-terminal regulatory domain is presented.     

Nerve growth factor action is mediated by cyclic AMP- and Ca+2/phospholipid-dependent protein kinases

Nerve growth factor (NGF) mediates the phosphorylation of tyrosine hydroxylase in PC12 cells on two distinct peptide fragments, separable by two-dimensional tryptic phosphopeptide mapping (phosphopeptides T1 and T3). Phorbol diester derivatives capable of activating Ca+2/phospholipid-dependent protein kinase (C-kinase) cause a specific phosphorylation of peptide T3 in a dose-dependent, saturable manner. Derivatives of the endogenous C-kinase activator diacylglycerol, also cause the phosphorylation of tyrosine hydroxylase on peptide T3. The C-kinase inhibitors chlorpromazine and trifluoperazine inhibit the phorbol diester stimulated phosphorylation of site T3 in a dose-dependent manner. These agents inhibit the phosphorylation of T3 in response to NGF, but have no effect on NGF's ability to cause T1 phosphorylation. In a PC12 mutant deficient in cAMP-dependent protein kinase activity, NGF mediates the phosphorylation of tyrosine hydroxylase on peptide T3 but not on T1. We conclude that NGF mediates the activation of both the cAMP-dependent protein kinase and the C-kinase to phosphorylate substrate proteins. These kinases can act independently to phosphorylate tyrosine hydroxylase, each at a different site, and each of which results in the enzyme activation. A molecular framework is thus provided for events underlying NGF action.     

The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains.

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.     

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.     

Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells.

Protein tyrosine phosphorylation has not been considered to be important for cellular activation by phospholipase C-linked vasoactive peptides. We found that endothelin, angiotensin II, and vasopressin (AVP), peptides that signal via phospholipase C activation, rapidly enhanced tyrosine phosphorylation of proteins of approximate molecular mass 225, 190, 135, 120, and 70 kDa in rat renal mesangial cells. The phosphorylated proteins were cytosolic or membrane-associated, and none were integral to the membrane, suggesting that the peptide receptors are not phosphorylated on tyrosine. Epidermal growth factor (EGF), which does not activate phospholipase C in these cells, induced the tyrosine phosphorylation of its own 175-kDa receptor, in addition to five proteins of identical molecular mass to those phosphorylated in response to endothelin, AVP, and angiotensin II. This suggests that in mesangial cells there is a common signaling pathway for phospholipase C-coupled agonists and agonists classically assumed to signal via receptor tyrosine kinase pathways, such as EGF. The phorbol ester, phorbol 12-myristate 13-acetate, and the synthetic diacylglycerol, oleoyl acetylglycerol, stimulated the tyrosine phosphorylation of proteins identical to those phosphorylated by the phospholipase C-linked peptides, suggesting that protein kinase C (PKC) activation is sufficient to active tyrosine phosphorylation. However, the PKC inhibitor, staurosporine, and down-regulation of PKC activity by prolonged exposure to phorbol esters completely inhibited tyrosine phosphorylation in response to PMA but not to endothelin, AVP, or EGF. In conclusion, endothelin, angiotensin II, and AVP enhances protein tyrosine phosphorylation via at least two pathways, PKC-dependent and PKC-independent. Although activation of PKC may be sufficient to enhance protein tyrosine phosphorylation, PKC is not necessary and may not be the primary route by which these agents act. At least one of these pathways is shared with the growth factor EGF, suggesting not only common intermediates in the signaling pathways for growth factors and vasoactive peptides but also perhaps common cellular tyrosine kinases which phosphorylate these intermediates.     

The multifunctional Ca2+/calmodulin-dependent protein kinase mediates Ca2+-dependent phosphorylation of tyrosine hydroxylase

Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.     

Tyrosine Hydroxylase Is Activated and Phosphorylated on Different Sites in Rat Pheochromocytoma PC12 Cells Treated with Phorbol Ester and Forskolin

Abstract: Incubation of rat pheochromocytoma PC12 cells with 4β-phorbol-12β-myristate-13α-acetate (PMA), an activator of Ca²⁺/phospholipid-dependent protein kinase (protein kinase C), or forskolin, an activator of adenylate cyclase, is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase. Neither the activation nor increased phosphorylation of tyrosine hydroxylase produced by PMA is dependent on extracellular Ca²⁺. Both activation and phosphorylation of the enzyme by PMA are inhibited by pretreatment of the cells with trifluo-perazine (TFP). Treatment of PC 12 cells with l-oleoyl-2-acetylglycerol also leads to increases in the phosphorylation and enzymatic activity of tyrosine hydroxylase; 1, 2-diolein and 1, 3-diolein are ineffective. The effects of forskolin on the activation and phosphorylation of the enzyme are independent of Ca²⁺ and are not inhibited by TIT⁵. Forskolin elicits an increase in cyclic AMP levels in PC 12 cells. The increases in both cyclic AMP content and the enzymatic activity and phosphorylation of tyrosine hydroxylase following exposure of PC 12 cells to different concentrations of forskolin are closely correlated. In contrast, cyclic AMP levels do not increase in cells treated with PMA. Tryptic digestion of the phosphorylated enzyme isolated from untreated cells yields four phosphopeptides separable by HPLC. Incubation of the cells in the presence of the Ca²⁺ ionophore ionomycin increases the phosphorylation of three of these tryptic peptides. However, in cells treated with either PMA or forskolin, there is an increase in the phosphorylation of only one of these peptides derived from tyrosine hydroxylase. The peptide phosphorylated in PMA-treated cells is different from that phosphorylated in forskolin-treated cells. The latter peptide is identical to the peptide phosphorylated in dibutyryl cyclic AMP-treated cells. These results indicate that tyrosine hydroxylase is activated and phosphorylated on different sites in PC 12 cells exposed to PMA and forskolin and that phosphorylation of either of these sites is associated with activation of tyrosine hydroxylase. The results further suggest that cyclic AMP-dependent and Ca²⁺/ phospholipid-dependent protein kinases may play a role in the regulation of tyrosine hydroxylase in PC 12 cells.     

Nerve Growth Factor (NGF) Induces a Rapid and Sustained Downregulation of the Focal Adhesion Kinase (FAK)

1. Exposure of PC12 cells to nerve growth factor (NGF) induces an early tyrosine phosphorylation of many proteins, a number of which is still unidentified. Although NGF is known to bind to and activate the receptor tyrosine kinase TrkA, many downstream targets of NGF signaling may be possibly phosphorylated by nonreceptor tyrosine kinases such as c-Src and focal adhesion kinase (FAK). 2. In the present study, exposure of TrkA-overexpressing PC12 cells to NGF is found to cause a rapid and sustained loss in the recovery of a subpopulation of nominally active FAK (i.e., being autophosphorylated on the positive site of regulation). 3. Consistent with the possibility that NGF induces the proteolysis of FAK via recruitment of Src family kinases, the use of various phosphorylation site-specific anti-FAK antibodies revealed an NGF-inducible and PP1-sensitive accumulation of a putative fragment (i.e., p62) of FAK. Significantly, the mitogenic epidermal growth factor (EGF) failed to induce the downregulation of FAK and the accumulation of tyrosine phosphorylated p62. Such differential response of FAK to NGF and EGF may shape the specificity by which these growth factors control the status of cell-matrix adhesion and the adhesion-driven signaling.     

Phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase. Temporal correlation of acetylcholine's effect on site phosphorylation, enzyme activation, and catecholamine synthesis

Tryptic peptide fragments of tyrosine hydroxylase isolated from 32PO4-prelabeled bovine adrenal chromaffin cells are resolved into seven phosphopeptides by reverse phase-high performance liquid chromatography. All seven of the peptides are phosphorylated on serine residues. Three of these putative phosphorylation sites, peptides 3, 5, and 6, are rapidly phosphorylated (5-fold in 15 s) by both acetylcholine stimulation and potassium depolarization of the cells, and this phosphorylation is accompanied by a similarly rapid activation of the enzyme. Both phosphorylation and activation are transient and do not account for the prolonged increase in catecholamine biosynthesis produced by these stimuli. Peptides 4 and 7 show a much slower and sustained increase in phosphorylation (3-fold in 4 min) in response to acetylcholine and potassium. Phosphorylation of these peptides correlates with the sustained increase in catecholamine biosynthesis rather than enzyme activation. Peptides 1 and 2 are not stimulated by any agonist yet employed and thus show no relation to enzyme activation or catecholamine biosynthesis. Phosphorylation of all five peptides by acetylcholine or potassium is calcium-dependent. In contrast to the stimulation of phosphorylation of tyrosine hydroxylase on multiple sites, forskolin stimulates the phosphorylation of only peptide 6, and this is accompanied by a coordinated activation of tyrosine hydroxylase and increased catecholamine biosynthesis. These findings show that the phosphorylation of tyrosine hydroxylase in intact cells is more complex than predicted from in vitro results, that at least two protein kinases are involved in the secretagogue-induced phosphorylation of tyrosine hydroxylase, and that the regulation of catecholamine biosynthesis, in response to phosphorylation, appears to involve both tyrosine hydroxylase activation and other mechanisms.     

Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid.

Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

Extracellular signal-regulated kinases 2 autophosphorylates on a subset of peptides phosphorylated in intact cells in response to insulin and nerve growth factor: analysis by peptide mapping.

The phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) in response to insulin in Rat 1 HIRc B cells and in response to nerve growth factor (NGF) in PC12 cells has been examined. ERK1 and ERK2 are phosphorylated on serine in the absence of the stimuli and additionally on tyrosine and threonine residues after exposure to NGF and insulin. NGF stimulates tyrosine phosphorylation of ERK1 more rapidly than threonine phosphorylation. Two-dimensional phosphopeptide maps of both ERK1 and ERK2 phosphorylated in intact cells treated with NGF or with insulin display the same three predominant phosphopeptides that comigrate when digests of ERK1 and ERK2 are mixed. As many as five additional phosphopeptides are detected under certain conditions. Autophosphorylated recombinant ERK2 also contains the three tryptic phosphopeptides found in ERKs labeled in intact cells. These experiments demonstrate that ERK1 and ERK2 are phosphorylated on related sites in response to two distinct extracellular signals. The data also support the possibility that autophosphorylation may be involved in the activation of the ERKs.     

cAMP-mediated modulation of signal transduction of epidermal growth factor (EGF) receptor systems in human epidermoid carcinoma A431 cells. Depression of EGF-dependent diacylglycerol production and EGF receptor phosphorylation.

While a cAMP-dependent protein kinase (protein kinase A) has been suggested to phosphorylate epidermal growth factor (EGF) receptor in vitro, both intrinsic and EGF- or potent phorbol tumor promoter-induced phosphorylation of EGF receptor were found to be depressed in human epidermoid carcinoma A431 cells by prior incubation of the cells with various protein kinase A activators (e.g. cholera toxin, forskolin, cAMP analogues, or a combination of prostaglandin E1 and 3-isobutyl-1-methylxanthine). Protein kinase A activators did not change significantly either the number of EGF receptors or their affinity for EGF. The tryptic phosphopeptide map of EGF receptors from cells treated with cholera toxin alone or cholera toxin followed by EGF revealed unique peptides whose serine phosphorylation was preferentially depressed. However, the catalytic subunit of protein kinase A phosphorylated no threonine and little serine in the EGF receptors in the plasma membranes of isolated A431 cells in vitro, while serine residues in an unidentified 170-kDa membrane protein(s) other than EGF receptor were heavily phosphorylated. Pretreatment of the cells with forskolin blocked 1,2-diacylglycerol induction by EGF; growth inhibition by nanomolar levels of EGF could be partially restored by the presence of forskolin. These results indicate that an increase in intracellular cAMP modulates the EGF receptor signal transduction system by reducing EGF-induced production of diacylglycerol without direct phosphorylation of EGF receptors by protein kinase A in A431 cells.     

Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C

Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.     

Phosphorylation of Tyrosine Hydroxylase by Cyclic GMP-Dependent Protein Kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated and activated by purified cyclic GMP-dependent protein kinase as well as by cyclic AMP-dependent protein kinase catalytic subunit. The extent of activation was correlated with the degree of phosphate incorporated into the enzyme. Comparable stoichiometric ratios (0.6 mol phosphate/mol tyrosine hydroxylase subunit) were obtained at maximal concentrations of either cyclic AMP-dependent or cyclic GMP-dependent protein kinases. The enzymes appeared to mediate the phosphorylation of the same residue based on the observation that incorporation was not increased when both enzymes were present. The major tryptic phosphopeptide obtained from tyrosine hydroxylase phosphorylated by each protein kinase exhibited an identical retention time following HPLC. The purified phosphopeptides also exhibited identical isoelectric points. These data provide support for the notion that the protein kinases are phosphorylating the same residue of tyrosine hydroxylase.     

Threonine 1336 of the human insulin receptor is a major target for phosphorylation by protein kinase C

The ability of tumor-promoting phorbol diesters to inhibit both insulin receptor tyrosine kinase activity and its intracellular signaling correlates with the phosphorylation of the insulin receptor beta subunit on serine and threonine residues. In the present studies, mouse 3T3 fibroblasts transfected with a human insulin receptor cDNA and expressing greater than one million of these receptors per cell were labeled with [32P]phosphate and treated with or without 100 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). Phosphorylated insulin receptors were immunoprecipitated and digested with trypsin. Alternatively, insulin receptors affinity purified from human term placenta were phosphorylated by protein kinase C prior to trypsin digestion of the 32P-labeled beta subunit. Analysis of the tryptic phosphopeptides from both the in vivo and in vitro labeled receptors by reversed-phase HPLC and two-dimensional thin-layer separation revealed that PMA and protein kinase C enhanced the phosphorylation of a peptide with identical chromatographic properties. Partial hydrolysis and radiosequence analysis of the phosphopeptide derived from insulin receptor phosphorylated by protein kinase C indicated that the phosphorylation of this tryptic peptide occurred specifically on a threonine, three amino acids from the amino terminus of the tryptic fragment. Comparison of these data with the known, deduced receptor sequence suggested that the receptor-derived tryptic phosphopeptide might be Ile-Leu-Thr(P)-Leu-Pro-Arg. Comigration of a phosphorylated synthetic peptide containing this sequence with the receptor-derived phosphopeptide confirmed the identity of the tryptic fragment. The phosphorylation site corresponds to threonine 1336 in the human insulin receptor beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of Microtubule-Associated Protein 2 at Distinct Sites by Calmodulin-Dependent and Cyclic-AMP-Dependent Kinases

Microtubule-associated protein 2 (MAP2) is an excellent substrate for both cyclic-AMP (cAMP)-dependent and Ca2+/calmodulin-dependent kinases. A recently purified cytosolic Ca2+/calmodulin-dependent kinase (now designated CaM kinase II) phosphorylates MAP2 as a major substrate. We now report that microtubule-associated cAMP-dependent and calmodulin-dependent protein kinases phosphorylate MAP2 on separate sites. Tryptic phosphopeptide digestion and two-dimensional phosphopeptide mapping revealed 11 major peptides phosphorylated by microtubule-associated cAMP-dependent kinase and five major peptide species phosphorylated by calmodulin-dependent kinase. All 11 of the cAMP-dependently phosphorylated peptides were phosphorylated on serine residues, whereas four of five major peptides phosphorylated by the calmodulin-dependent kinase were phosphorylated on threonine. Only one peptide spot phosphorylated by both kinases was indistinguishable by both migration and phosphoamino acid site. The results indicate that cAMP-dependent and calmodulin-dependent kinases may regulate microtubule and cytoskeletal dynamics by phosphorylation of MAP2 at distinct sites.     

Synergistic modulation of ectoCa2(+)-ATPase activity of hepatoma (Li-7A) cells by epidermal growth factor and cyclic AMP

A mercurial-insensitive ectoATPase, which was more active with CaATP than with MgATP, was induced when human hepatoma (Li-7A) cells were cultured in the presence of epidermal growth factor (EGF) and cholera toxin. Cholera toxin could be replaced by forskolin, 8-Br-cAMP, butyryl-cAMP, and dibutyryl-cAMP. Requirement for EGF was specific, but EGF was ineffective if added more than 24 h after the addition of forskolin or cholera toxin. It was concluded that induction of the ectoCa2(+)-ATPase was a consequence of the synergistic actions of EGF and cyclic AMP. The tyrosine kinase activity of the EGF receptor was essential for the induction of ectoCa2(+)-ATPase, since enzyme induction was abolished by a tyrosine kinase inhibitor, genistein. Cycloheximide and actinomycin D were also inhibitory to enzyme induction, indicating that enhancement of enzyme activity by EGF and cAMP was not due to post-translational modification. The results of this and previous investigations established that the two ectoATPases of Li-7A cells are under different regulation.