Tyrosine kinase activity of insulin receptors from human placenta. Effects of autophosphorylation and cyclic AMP-dependent protein kinase.

The kinase activity of partially purified insulin receptor obtained from human placenta was studied. When autophosphorylation of the beta-subunit of the receptor was initiated by ATP prior to the addition of the exogenous substrate, both basal and insulin-stimulated kinase activity was increased. However, half-maximum effective insulin concentrations were unchanged. Insulin receptor autophosphorylation as stimulated by ATP and insulin failed to affect significantly 125I-insulin binding to partially purified insulin receptor from human placenta. It is concluded that autophosphorylation of the insulin receptors regulates its kinase activity but not its affinity for insulin. The catalytic subunit of cyclic AMP-dependent protein kinase failed to phosphorylate either subunit of the insulin receptor, and each kinase failed to affect the affinity of the other one. Thus no functional interaction between cyclic AMP-dependent protein kinase and insulin receptors was observed in the in vitro system.     

14-3-3 binding to the IGF-1 receptor is mediated by serine autophosphorylation

The phosphoserine-binding 14-3-3 proteins have been implicated in playing a role in mitogenic and apoptotic signaling pathways. Binding of 14-3-3 proteins to phosphoserine residues in the C-terminus of the insulin-like growth factor-1 receptor (IGF-1R) has been described to occur in a variety of cell systems, but the kinase responsible for this serine phosphorylation has not been identified yet. Here we present evidence that the isolated dimeric insulin-like growth factor-1 receptor kinase domain (IGFKD) contains a dual specific (i.e. tyrosine/serine) kinase activity that mediates autophosphorylation of C-terminal serine residues in the enzyme. From the total phosphate incorporation of approximately 4 mol per mol kinase subunit, 1 mol accounts for serine phosphate. However, tyrosine autophosphorylation proceeds more rapidly than autophosphorylation of serine residues (t(1/2) approximately 1 min vs. t(1/2) approximately 5 min). Moreover, dot-blot and far-Western analyses reveal that serine autophosphorylation of IGFKD is sufficient to promote binding of 14-3-3 proteins in vitro. The proof that dual kinase activity of IGFKD is necessary and sufficient for 14-3-3 binding was obtained with an inactive kinase mutant that was phosphorylated on serine residues in a stoichiometric reaction with the catalytically active enzyme. Thus, the IGF-1R itself might be responsible for the serine autophosphorylation which leads to recognition of 14-3-3 proteins in vivo.     

The 93 kDa protein gephyrin and tubulin associated with the inhibitory glycine receptor are phosphorylated by an endogenous protein kinase.

The 93 kDa protein gephyrin is a tubulin binding peripheral membrane protein that is associated with the inhibitory glycine receptor and has been implicated in its anchoring at central synapses. Here, we demonstrate that gephyrin as well as co-purifying tubulin are phosphorylated by a kinase activity which is endogenous to highly purified glycine receptor preparations. This kinase phosphorylates serine and threonine residues and utilizes ATP, but not GTP, as phosphate donor. Its activity is not affected by various activators and/or inhibitors of cyclic nucleotide-dependent kinases, calcium/calmodulin-dependent kinases, or protein kinase C. A five-fold stimulation of kinase activity was, however, observed in the presence of poly-lysine. Phosphorylation of gephyrin and/or tubulin might regulate receptor/cytoskeleton interactions at postsynaptic membrane specializations.     

Global Forebrain Ischemia Induces a Posttranslational Modification of Multifunctional Calcium- and Calmodulin-Dependent Kinase II

The activity of multifunctional calcium/calmodulin-dependent protein kinase II (CaM kinase II) has recently been shown to be inhibited by transient global ischemia. To investigate the nature of ischemia-induced inhibition of the enzyme, CaM kinase II was purified to greater than 1,000-fold from brains of control and ischemic gerbils. The characteristics of CaM kinase II from control and ischemic preparations were compared by numerous parameters. Kinetic analysis of purified control and ischemic CaM kinase II was performed for autophosphorylation properties, ATP, magnesium, calcium, and calmodulin affinity, immunoreactivity, and substrate recognition. Ischemia induced a reproducible inhibition of CaM kinase II activity, which could not be overcome by increasing the concentration of any of the reaction parameters. Ischemic CaM kinase II was not different from control enzyme in affinity for calmodulin, Ca2+, Mg2+, or exogenously added substrate or rate of autophosphorylation. CaM kinase II isolated from ischemic gerbils displayed decreased immunoreactivity with a monoclonal antibody (immunoglobulin G3) directed toward the beta subunit of the enzyme. In addition, ischemia caused a significant decrease in affinity of CaM kinase II for ATP when measured by extent of autophosphorylation. To characterize further the decrease in ATP affinity of CaM kinase II, the covalent-binding ATP analog 8-azido-adenosine-5'-[alpha-32P]triphosphate was used. Covalent binding of 25 microM azido-ATP was decreased 40.4 +/-12.3% in ischemic CaM kinase II when compared with control enzyme (n = 5; p less than 0.01 by paired Student's t test). Thus, CaM kinase II levels for ischemia and control fractions were equivalent by protein staining, percent recovery, and calmodulin binding but were significantly different by immunoreactivity and ATP binding. The data are consistent with the hypothesis that ischemia induces a posttranslational modification that alters ATP binding in CaM kinase II and that results in an apparent decrease in enzymatic activity.     

Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation

Epidermal growth factor (EGF) receptor protein has been purified in a single high-yield step by immunoaffinity chromatography of extracts of A431 cells. A monoclonal antibody directed against the EGF binding site of the receptor was immobilized to Sepharose 4B as a specific immune absorbent and competitive elution with EGF was used to obtain purified EGF receptor protein with tyrosine kinase activity. The stoichiometry of EGF binding was determined by comparing 125I-EGF binding to A431 cells with the mass of EGF receptor protein in those cells as measured by immunoaffinity chromatography, radioimmunoassay, and immune precipitation. Each measurement indicated one EGF binding site/EGF receptor protein molecule. Study of the kinetics of autophosphorylation revealed rapid incorporation of 1 mol of phosphate/mol of enzyme followed by slower incorporation of additional phosphate groups. The autophosphorylation reaction has a Km for ATP (0.2 microM) which is about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation is an intramolecular reaction.     

A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine.

Platelet-derived growth factor (PDGF) stimulates autophosphorylation of the PDGF receptor and association of the receptor with several cytoplasmic molecules, including phosphatidylinositol-3 kinase (PI3 kinase). In this study we examined the association of PI3 kinase with immunoprecipitated autophosphorylated PDGF receptor in vitro. The PI3 kinase from cell lysates bound to the wild-type receptor but not to a mutant receptor that had a deletion of the kinase insert region. A protein of an apparent size of 85 kDa bound to the receptor, consistent with previous observations that a protein of this size is associated with PI3 kinase activity. In addition, 110- and 74-kDa proteins bound to the phosphorylated receptor. Dephosphorylated receptors lost the ability to bind PI3 kinase activity as well as the 85-kDa protein. A 20-amino-acid peptide composed of a sequence in the kinase insert region that included one of the autophosphorylation sites of the receptor (tyrosine 719) as well as a nearby tyrosine (Y708) blocked the binding of PI3 kinase to the receptor, but only when the peptide was phosphorylated on tyrosine residues. A scrambled version of the peptide did not block PI3 kinase binding to the receptor even when it was phosphorylated on tyrosine. These tyrosine-phosphorylated peptides did not block binding of phospholipase C-gamma or GTPase-activating protein to the receptor. In separate experiments (receptor blots), soluble radiolabeled receptor bound specifically to an 85-kDa protein present in sodium dodecyl sulfate-polyacrylamide gel electrophoresis-fractionated 3T3 cell lysates that were transferred to nitrocellulose paper. The binding was blocked by the same tyrosine-phosphorylated peptides that prevented binding of PI3 kinase activity to immobilized receptors. These findings show that the PDGF receptor binds directly to an 85-kDa protein and to a PI3 kinase activity through specific sequences in the kinase insert region. The association of a 110-kDa protein with the receptor also involve these sequences, suggesting that this protein may be a subunit of the PI3 kinase. Phosphotyrosine is an essential structure required for the interactions of these proteins with the PDGF receptor.     

Effect of cyclic AMP-dependent protein kinase on insulin receptor tyrosine kinase activity.

To explain the insulin resistance induced by catecholamines, we studied the tyrosine kinase activity of insulin receptors in a state characterized by elevated noradrenaline concentrations in vivo, i.e. cold-acclimation. Insulin receptors were partially purified from brown adipose tissue of 3-week- or 48 h-cold-acclimated mice. Insulin-stimulated receptor autophosphorylation and tyrosine kinase activity of insulin receptors prepared from cold-acclimated mice were decreased. Since the effect of noradrenaline is mediated by cyclic AMP and cyclic AMP-dependent protein kinase, we tested the effect of the purified catalytic subunit of this enzyme on insulin receptors purified by wheat-germ agglutinin chromatography. The catalytic subunit had no effect on basal phosphorylation, but completely inhibited the insulin-stimulated receptor phosphorylation. Similarly, receptor kinase activity towards exogenous substrates such as histone or a tyrosine-containing copolymer was abolished. This inhibitory effect was observed with receptors prepared from brown adipose tissue, isolated hepatocytes and skeletal muscle. The same results were obtained on epidermal-growth-factor receptors. Further, the catalytic subunit exerted a comparable effect on the phosphorylation of highly purified insulin receptors. To explain this inhibition, we were able to rule out the following phenomena: a change in insulin binding, a change in the Km of the enzyme for ATP, activation of a phosphatase activity present in the insulin-receptor preparation, depletion of ATP, and phosphorylation of a serine residue of the receptor. These results suggest that the alteration in the insulin-receptor tyrosine kinase activity induced by cyclic AMP-dependent protein kinase could contribute to the insulin resistance produced by catecholamines.     

Increased protein kinase C activity is linked to reduced insulin receptor autophosphorylation in liver of starved rats

Phosphorylation of the insulin receptor beta-subunit on serine/threonine residues by protein kinase C reduces both receptor kinase activity and insulin action in cultured cells. Whether this mechanism regulates insulin action in intact animals was investigated in rats rendered insulin-resistant by 3 days of starvation. Insulin-stimulated autophosphorylation of the partially purified hepatic insulin receptor beta-subunit was decreased by 45% in starved animals compared to fed controls. This autophosphorylation defect was entirely reversed by removal of pre-existing phosphate from the receptor with alkaline phosphatase, suggesting that increased basal phosphorylation on serine/threonine residues may cause the decreased receptor tyrosine kinase activity. Tryptic removal of a C-terminal region of the receptor beta-subunit containing the Ser/Thr phosphorylation sites similarly normalized receptor autophosphorylation. To investigate which kinase(s) may be responsible for such increased Ser/Thr phosphorylation in vivo, protein kinase C and cAMP-dependent protein kinase A in liver were studied. A 2-fold increase in protein kinase C activity was found in both cytosol and membrane extracts from starved rats as compared to controls, while protein kinase A activity was diminished in the cytosol of starved rats. A parallel increase in protein kinase C was demonstrated by immunoblotting with a polyclonal antibody which recognizes several protein kinase C isoforms. These findings suggest that in starved, insulin-resistant animals, an increase in hepatic protein kinase C activity is associated with increased Ser/Thr phosphorylation which in turn decreases autophosphorylation and function of the insulin receptor kinase.     

Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.     

Autophosphorylation of neuronal calcium/calmodulin-stimulated protein kinase II

A unique feature of neuronal calcium/calmodulin-stimulated protein kinase II (CaM-PK II) is its autophosphorylation. A number of sites are involved and, depending on the in vitro conditions used, three serine and six threonine residues have been tentatively identified as autophosphorylation sites in the alpha subunit. These sites fall into three categories. Primary sites are phosphorylated in the presence of calcium and calmodulin, but under limiting conditions of temperature, ATP, Mg2+, or time. Secondary sites are phosphorylated in the presence of calcium and calmodulin under nonlimiting conditions. Autonomous sites are phosphorylated in the absence of calcium and calmodulin after initial phosphorylation of Thr-286. Mechanisms that lead to a decrease in CaM-PK II autophosphorylation include the thermolability of the enzyme and the activity of protein phosphatases. A range of in vitro inhibitors of CaM-PK II autophosphorylation have recently been identified. Autophosphorylation of CaM-PK II leads to a number of consequences in vitro, including generation of autonomous activity and subcellular redistribution, as well as alterations in conformation, activity, calmodulin binding, substrate specificity, and susceptibility to proteolysis. It is established that CaM-PK II is autophos-phorylated in neuronal cells under basal conditions. Depolarization and/or activation of receptors that lead to an increase in intracellular calcium induces a marked rise in the autophosphorylation of CaM-PK II in situ. The incorporation of phosphate is mainly found on Thr-286, but other sites are also phosphorylated at a slower rate. One consequence of the increase in CaM-PK II autophosphorylation in situ is an increase in the level of autonomous kinase activity. It is proposed that the formation of an autonomous enzyme is only one of the consequences of CaM-PK II autophosphorylation in situ and that some of the other consequences observed in vitro will also be seen. CaM-PK II is involved in the control of neuronal plasticity, including neurotransmitter release and long-term modulation of postreceptor events. In order to understand the function of CaM-PK II, it will be essential to ascertain more fully the mechanisms of its autophosphorylation in situ, including especially the sites involved, the consequences of this autophosphorylation for the kinase activity, and the relationships between the state of CaM-PK II autophosphorylation and the physiological events within neurons.     

Identification of inhibitory autophosphorylation sites in casein kinase I epsilon.

Casein kinase I epsilon (CKIepsilon) is a widely expressed protein kinase implicated in the regulation of diverse cellular processes including DNA replication and repair, nuclear trafficking, and circadian rhythm. CKIepsilon and the closely related CKIdelta are regulated in part through autophosphorylation of their carboxyl-terminal extensions, resulting in down-regulation of enzyme activity. Treatment of CKIepsilon with any of several serine/threonine phosphatases causes a marked increase in kinase activity that is self-limited. To identify the sites of inhibitory autophosphorylation, a series of carboxyl-terminal deletion mutants was constructed by site-directed mutagenesis. Truncations that eliminated specific phosphopeptides present in the wild-type kinase were used to guide construction of specific serine/threonine to alanine mutants. Amino acids Ser-323, Thr-325, Thr-334, Thr-337, Ser-368, Ser-405, Thr-407, and Ser-408 in the carboxyl-terminal tail of CKIepsilon were identified as probable in vivo autophosphorylation sites. A recombinant CKIepsilon protein with serine and threonine to alanine mutations eliminating these autophosphorylation sites was 8-fold more active than wild-type CKIepsilon using IkappaBalpha as a substrate. The identified autophosphorylation sites do not conform to CKI substrate motifs identified in peptide substrates.     

Pertussis toxin inhibits autophosphorylation and activation of the insulin receptor kinase.

Pertussis toxin is an ADP-ribosyltransferase which alters the function of some of the GTP-binding proteins and inhibits some actions of insulin. In vivo, pertussis toxin (2 micrograms/ml/2h) inhibited insulin-stimulated tyrosyl autophosphorylation of the insulin receptor by 50% in FaO cells, and nearly completely inhibited phosphorylation of the cellular insulin receptor substrate pp185. Similarly, insulin-stimulated autophosphorylation and kinase activity of the insulin receptor purified on wheat germ agglutinin-agarose from pertussis toxin-treated FaO cells was diminished 50%; however, treatment of cells with the catalytically inactive B-oligomer of the toxin had no effect on receptor tyrosine kinase activity in vitro. Pertussis toxin did not alter insulin binding or the cellular levels of ATP, cAMP, and cGMP. Furthermore, immunoprecipitation of the insulin receptor from intact cells with anti-insulin receptor antibodies showed that pertussis toxin did not increase the phosphorylation of serine or threonine residues in the insulin receptor. These results suggest that pertussis toxin can modulate signal transduction of insulin at the level of the insulin receptor kinase.     

Autophosphorylation of rat brain Ca2+-activated and phospholipid-dependent protein kinase

Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) isolated from rat brain cytosol undergoes autophosphorylation in the presence of Mg2+, ATP, Ca2+, phosphatidylserine, and diolein. Approximately 2-2.5 mol of phosphate were incorporated per mol of the kinase. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, the phosphorylated kinase showed a single protein band of Mr = 82,000 compared to the Mr = 80,000 of the nonphosphorylated enzyme. Analysis of the 32P-labeled tryptic peptides derived from the autophosphorylated kinase by peptide mapping revealed that multiple sites were phosphorylated. Both serine and threonine residues were found to be labeled with 32P. Limited proteolysis of the autophosphorylated kinase with trypsin resulted in the conversion of the kinase into a phospholipid- and Ca2+-independent form. Two major 32P-labeled fragments, Mr = 48,000 and 38,000, were formed as a result of proteolysis, suggesting that the catalytic domain and possibly the Ca2+- and phospholipid-binding region were both phosphorylated. Protein kinase C autophosphorylation has a Km for ATP (1.5 microM) about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation appears to be an intramolecular reaction. The autophosphorylated protein kinase C, unlike the protease-degraded enzyme, still depends on Ca2+ and phospholipid for maximal activity. However, the autophosphorylated form of the kinase has a lower Ka for Ca2+ and a higher affinity for the binding of [3H]phorbol-12, 13-dibutyrate. These findings suggest that autophosphorylation of protein kinase C may be important in the regulation of the enzymic activity subsequent to signal transduction.     

The 90-kDa heat shock protein (hsp-90) possesses an ATP binding site and autophosphorylating activity

The 90-kDa heat shock protein (hsp-90) is an abundant cytosolic protein believed to play a role in maintenance of protein trafficking and closely associated with several steroid hormone receptors. Incubation of highly purified hsp-90 with [gamma-32P]ATP results in its autophosphorylation on serine residues. There are several lines of evidence which suggest that this activity is due to a kinase intrinsic to hsp-90 rather than some closely associated protein kinases: 1) the phosphorylation persists after the removal of casein kinase II by heparin chromatography and after immunoprecipitation of hsp-90 with anti-hsp-90 antibodies. 2) The approximate kM for ATP of the reaction is 0.16 mM, higher than that of many other protein kinases. 3) Phosphorylation is not affected by a number of activators and inhibitors of other known kinases which might associate with hsp-90. 4) The phosphorylation displays a unique cation dependence being most active in the presence of Ca2+ and practically inactive with Mg2+, although the autophosphorylation in the presence of Mg2+ is activated by histones and polyamines. 5) The activity is remarkably heat-stable; incubation of hsp-90 for 20 min at 95 degrees C results in only a 60% decrease in autophosphorylation. 6) Finally, and most importantly, purified hsp-90 can be labeled with azido-ATP and it is able to bind to ATP-agarose. The binding shows similar cation dependence to the autophosphorylation. These data are in agreement with the presence of a consensus sequence for ATP binding sites in the primary structure of the protein similar to that observed in the 70-kDa heat-shock proteins. Our data suggest the 90-kDa heat shock protein possesses an enzymatic activity analogous in many respects to the similar activity of the 70-kDa heat shock proteins. This may represent an important, previously unrecognized function of hsp-90.     

A synthetic peptide corresponding to residues 137 to 157 of p60v-src inhibits tyrosine-specific protein kinases

A 21-residue synthetic peptide corresponding to a part of the noncatalytic domain of p60v-src (residues 137 to 157) was found to inhibit the tyrosine kinase activity of p60v-src. The half inhibition concentration was ca. 7.5 microM. The peptide (peptide A) did not compete with substrate proteins or ATP. Peptide A also inhibited the autophosphorylation of epidermal growth factor receptor/kinase and the tyrosine-specific protein phosphorylation in the acetylcholine receptor-rich membranes isolated from electroplax of Narke japonica. However, serine/threonine-specific protein kinases such as cAMP-dependent and cGMP-dependent protein kinases were not inhibited by peptide A.     

Vascular endothelial growth factor receptor KDR tyrosine kinase activity is increased by autophosphorylation of two activation loop tyrosine residues

Vascular endothelial growth factor is an important physiological regulator of angiogenesis. The function of this endothelial cell selective growth factor is mediated by two homologous tyrosine kinase receptors, fms-like tyrosine kinase 1 (Flt-1) and kinase domain receptor (KDR). Although the functional consequence of vascular endothelial growth factor binding to the Flt-1 receptor is not fully understood, it is well established that mitogenic signaling is mediated by KDR. Upon sequencing several independent cDNA clones spanning the cytoplasmic region of human KDR, we identified and confirmed the identity of a functionally required valine at position 848 in the ATP binding site, rather than the previously reported glutamic acid residue, which corresponds to an inactive tyrosine kinase. The cytoplasmic domain of recombinant native KDR, expressed as a glutathione S-transferase fusion protein, can undergo autophosphorylation in the presence of ATP. In addition, the kinase activity can be substantially increased by autophosphorylation at physiologic ATP concentrations. Mutation analysis indicates that both tyrosine residues 1054 and 1059 are required for activation, which is a consequence of an increased affinity for both ATP and the peptide substrate and has no effect on kcat, the intrinsic catalytic activity of the enzyme. KDR kinase catalyzes phosphotransfer by formation of a ternary complex with ATP and the peptide substrate. We demonstrate that tyrosine kinase antagonists can preferentially inhibit either the unactivated or activated form of the enzyme.     

Autophosphorylation of EGF receptors in the A431 cell line with an increased intracellular concentration of Ca(2+) ions

The increase in the intracellular concentration of Ca2+ in A431 cells induced by the calcium ionophore A23187 leads to phosphorylation of epidermal growth factor (EGF) receptors at serine and/or threonine residues. This process is accompanied by the decrease in the level of EGF receptor autophosphorylation at tyrosine residues. Preincubation of cells in a A23187-containing medium in the presence of phorbol-12-myristoyl-13-acetate leads to a further decrease of the phosphotyrosine content in EGF receptors. At increased intracellular concentrations of Ca2+ preincubation of A431 cells with the protein kinase C inhibitor H-7 has no effect on the degree of EGF receptor autophosphorylation. Down-regulation of cellular protein kinase C does not change the A23187-induced effect either. The data obtained suggest that the decreased autophosphorylation of EGF receptors induced by Ca2+ is not due to the activation of cellular protein kinase C.     

Kinetic properties of the insulin receptor tyrosine protein kinase: activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation

The insulin receptor is an insulin-activated, tyrosine-specific protein kinase. Previous studies have shown that autophosphorylation of tyrosine residues on the Mr 95,000 is associated with an activation of the protein kinase activity toward exogenous protein substrates. We have employed the highly purified insulin receptor, immobilized on insulin-Sepharose or eluted in an active form, to define the metal/ATP requirements for kinase activation, the relationship of receptor autophosphorylation to activation, and the kinetic properties of the autophosphorylated, activated receptor kinase. Prior incubation of the immobilized receptor with 2 mM ATP, 10 mM Mg (or 10 mM Mn), followed by removal of these reactants, served to abolish the upward curvilinearity in the rate of histone 2b (tyrosine) phosphorylation measured subsequently. This treatment also markedly increased the rate of histone 2b phosphorylation as compared to that observed with the unmodified, immobilized receptor, as estimated under conditions that per se minimized further activation. The extents of maximal activation of receptor histone 2b (tyrosine) kinase obtained on preincubation with MgATP or MnATP are identical; however, the affinity of the receptor for MnATP is approximately 10-fold higher than that for MgATP. The higher affinity of the receptor for MnATP is observed for both autophosphorylation/autoactivation and histone 2b tyrosine kinase activity (Km MnATP approximately 0.01 mM; Km MgATP approximately 0.1 mM). Autophosphorylation/autoactivation per se does not significantly alter the apparent affinity for MeATP (or protein substrate, as previously reported) but increases Vmax. Activation of receptor histone 2b (tyrosine) kinase is due to tyrosine-specific autophosphorylation of the Mr 95,000 (beta) subunit; thus the extent of total 32P incorporation into the beta subunit correlates precisely with the extent of kinase activation, both over time and at a wide variety of Me2+ ATP concentrations. Sequential treatment of the autophosphorylated receptor with elastase and trypsin yields a single, basically charged 32P-peptide, Mr less than 2000. The functional properties of the unphosphorylated and fully phosphorylated receptor were compared after elution from insulin-Sepharose. The insulin binding characteristics of the two forms of the receptor were indistinguishable; the kinase properties differed greatly; whereas the histone 2b activity of the unphosphorylated receptor was low in the basal state, and activated 10-fold by insulin, the fully autophosphorylated receptor exhibits maximal histone 2b kinase in the basal state and is unaffected by insulin addition.(ABSTRACT TRUNCATED AT 400 WORDS)     

Involvement of protein kinase D in expression and trafficking of ATP7B (copper ATPase)

ATP7B is a P-type ATPase involved in copper transport and homeostasis. In experiments with microsomes isolated from COS-1 cells or HepG2 hepatocytes sustaining ATP7B heterologous expression, we found that ATP7B utilization of ATP includes autophosphorylation of an aspartyl residue serving as ATPase catalytic intermediate as well as phosphorylation of serine residues by protein kinase D (PKD). The latter was abolished by specific PKD inhibition with CID755673. The presence of PKD protein in the microsomal fraction was demonstrated by Western blotting. PKD is a serine/threonine kinase that associates with the trans-Golgi network, regulating fission of transport carriers destined to the cell surface. Parallel studies on cultured cells showed that nascent WT ATP7B transits to the Golgi complex where it undergoes serine phosphorylation by PKD. Misfolded ATP7B protein (especially if subjected to deletions) underwent proteasome-mediated degradation, which provides effective quality control. Inhibition of proteasome-mediated degradation with MG132 yielded additional, but nonfunctional protein. On the other hand, serine phosphorylation protected WT ATP7B from degradation. Protection was enhanced by PKD activation with phorbol esters and limited by PKD inhibition with CID75673. As a final step, phosphorylated ATP7B was transferred from the Golgi complex to cytosolic trafficking vesicles. Phosphorylation and trafficking were completely prevented by mutations of critical copper binding sites, demonstrating copper dependence of both PKD-assisted phosphorylation and trafficking. ATP7B trafficking was markedly reduced by the Ser-478/481/1121/1453 to Ala mutation. We conclude that PKD plays a key role in copper-dependent serine phosphorylation, permitting high levels of ATP7B protein expression and trafficking.