Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.     

Autophosphorylation of gatekeeper tyrosine by symbiosis receptor kinase

Plant receptor-like kinases (RLKs) share their evolutionary origin with animal interleukin-1 receptor-associated kinase (IRAK)/Pelle family of soluble kinases and are distinguished by having tyrosine as ‘gatekeeper’. This position is adjacent to the hinge region and is hidden in a hydrophobic pocket of the catalytic cleft of protein kinases and is therefore least probable to be a target for any modification. This communication illustrates the accessibility of the gatekeeper site (Y670) towards both autophosphorylation and dephosphorylation in the recombinant cytoplasmic domain of symbiosis receptor kinase from Arachis hypogaea (AhSYMRK). Autophosphorylation on gatekeeper tyrosine was detected prior to extraction but never under in vitro conditions. We hypothesize gatekeeper phosphorylation to be associated with synthesis/maturation of AhSYMRK and this phenomenon may be prevalent among RLKs.     

Characterization of fodrin phosphorylation by spleen protein tyrosine kinase

Fodrin, an actin and calmodulin binding and spectrin-like protein present in many nonerythrocyte tissues, could be phosphorylated up to more than 1.5 mol of phosphate/mol of protein by a highly purified non-receptor-associated protein tyrosine kinase from bovine spleen. The protein phosphorylation was not affected by Ca2+/calmodulin or by F-actin. Km and Vmax values of the reaction were 91 nM and 0.35 nmol of P2 min-1 (mg of kinase)-1, respectively. Both subunits A and B of fodrin were phosphorylated, with the rate of subunit A phosphorylation much greater than that of subunit B phosphorylation. Tryptic phosphopeptide mapping of the phosphorylated subunits suggested that there were three major phosphorylation sites in subunit A and one in subunit B. Phosphotyrosylfodrin could be dephosphorylated by the calmodulin-stimulated phosphatase (calcineurin) in the presence of activating metal ions; Ni2+ was a much more effective activator than Mn2+ for this reaction. Fodrin phosphorylation by the spleen protein tyrosine kinase did not appear to alter the actin and calmodulin binding properties of the protein. On the other hand, the calmodulin-dependent stimulation of smooth muscle actomyosin Mg2+-ATPase by fodrin was enhanced by 101% +/- 3% (n = 3) upon fodrin phosphorylation. Ni2+-calcineurin, which was shown to effectively dephosphorylate the phosphotyrosyl residues on fodrin, could reverse the phosphorylation-enhanced Mg2+-ATPase stimulatory activity of fodrin.     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Autophosphorylation of cGMP-dependent protein kinase type II

Cyclic nucleotides are shown to stimulate the autophosphorylation of type II cGMP-dependent protein kinase (cGK) on multiple sites. Mass spectrometric based analyses, using a quadrupole time-of-flight-mass spectrometry instrument revealed that cGMP stimulated the in vitro phosphorylation of residues Ser110 and Ser114, and, at a slow rate, of Ser126 and Thr109 or Ser117, all located in the autoinhibitory region. In addition Ser445 was found to be phosphorylated in a cGMP-dependent manner, whereas Ser110 and Ser97 were already prephosphorylated to a large extent in Sf9 cells. cGMP-dependent phosphorylation of cGK II was also demonstrated in intact COS-1 cells and intestinal epithelium. Substitution of most of the potentially autophosphorylated residues for alanines largely abolished the cGMP stimulation of the autophosphorylation. Prolonged autophosphorylation of purified recombinant cGK II in vitro resulted in a 40-50% increase in basal kinase activity, but its maximal cGMP-stimulated activity and the EC50 for cGMP remained unaltered. Mutation of the major phosphorylatable serines 110, 114, and 445 into "phosphorylation-mimicking" glutamates had no effect on the kinetic parameters of cGK II. However, replacing the slowly autophosphorylated residue Ser126 by Glu rendered cGK II constitutively active. These results show that the fast phase of cyclic nucleotide-stimulated autophosphorylation of cGK II has a relatively small feed forward effect on its activity, whereas the secondary phase, presumably involving Ser126 phosphorylation, may generate a constitutively active form of the enzyme.     

Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity

Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.     

Autophosphorylation and protein kinase C phosphorylation of the epidermal growth factor receptor. Effect on tyrosine kinase activity and ligand binding affinity

The effect of autophosphorylation and protein kinase C-catalyzed phosphorylation on the tyrosine-protein kinase activity and ligand binding affinity of the epidermal growth factor (EGF) receptor has been studied. Kinetic parameters for the phosphorylation by the receptor kinase of synthetic peptide substrates having sequences related to the 3 in vitro receptor autophosphorylation sites (tyrosine residues 1173 (P1), 1148 (P2), and 1068 (P3)) were measured. The Km of peptide P1 (residues 1164-1176) was significantly lower than that for peptides P2 (residues 1141-1151) or P3 (residues 1059-1072). The tyrosine residue 1173 was also the most rapidly autophosphorylated in purified receptor preparations, consistent with previous observations for the receptor in intact cells (Downward, J., Parker, P., and Waterfield, M. D. (1984) Nature 311, 483-485). Variation in the extent of receptor autophosphorylation from 0.1 to 2.8 mol of phosphate/mol of receptor did not influence kinase activity or EGF binding affinity either for purified receptor or receptor in membrane preparations. Phosphorylation of the EGF receptor by protein kinase C was shown to cause a 3-fold decrease in the affinity of purified EGF receptor for EGF and to reduce the receptor kinase activity. In membrane preparations, phosphorylation of the EGF receptor by protein kinase C resulted in conversion of high affinity EGF binding sites to a low affinity state. This suggests that activation of protein kinase C by certain growth promoting agents and tumor promoters is directly responsible for modulation of the affinity of the EGF receptor for its ligand EGF. The regulation of the EGF receptor function by protein kinase C is discussed.     

Autophosphorylation of the Escherichia coli protein kinase Wzc regulates tyrosine phosphorylation of Ugd,a UDP-glucose dehydrogenase

Autophosphorylation of protein-tyrosine kinases (PTKs) involved in exopolysaccharide and capsular polysaccharide biosynthesis and transport has been observed in a number of Gram-negative and Gram-positive bacteria. However, besides their own phosphorylation, little is known about other substrates targeted by these protein-modifying enzymes. Here, we present evidence that the protein-tyrosine kinase Wzc of Escherichia coli is able to phosphorylate an endogenous enzyme, UDP-glucose dehydrogenase (Ugd), which participates in the synthesis of the exopolysaccharide colanic acid. The process of phosphorylation of Ugd by Wzc was shown to be stimulated by previous autophosphorylation of Wzc on tyrosine 569. The phosphorylation of Ugd was demonstrated to actually occur on tyrosine and result in a significant increase of its dehydrogenase activity. In addition, the phosphotyrosine-protein phosphatase Wzb, which is known to effectively dephosphorylate Wzc, exhibited only a low effect, if any, on the dephosphorylation of Ugd. These data were related to the recent observation that two other UDP-glucose dehydrogenases have been also shown to be phosphorylated by a PTK in the Gram-positive bacterium Bacillus subtilis. Comparative analysis of the activities of PTKs from Gram-negative and Gram-positive bacteria showed that they are regulated by different mechanisms that involve, respectively, either the autophosphorylation of kinases or their interaction with a membrane protein activator.     

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.     

Autophosphorylation of rat liver type II cAMP-dependent protein kinase

A monoclonal antibody was prepared against the regulatory subunit (RII) of rat liver type II cAMP-dependent protein kinase. Autophosphorylated and nonphosphorylated RII in extracts from rat liver or hepatocytes were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and quantified by immunoblot analysis with this antibody. Under basal conditions, 90% of hepatocyte RII was in the phosphorylated form. Incubating hepatocytes with 8-bromo-cAMP and a phosphodiesterase inhibitor resulted in activation of cAMP-dependent protein kinase and glycogenolysis but did not affect phospho RII levels. RII phosphorylation was also unaffected by the inclusion of sufficient insulin to cause a decrease in cAMP-dependent protein kinase activity and glycogenolysis. The results indicate that unlike other cell types, dissociation of rat hepatocyte type II cAMP-dependent protein kinase does not result in dephosphorylation of RII. The biochemical basis for the apparent lack of RII dephosphorylation in intact hepatocytes was examined by comparison with smooth muscle where RII is rapidly dephosphorylated. Rat liver extract contained 4-fold less RII and had an 80-fold lower rate of dephosphorylation of endogenous RII compared to bovine smooth muscle extract. The differences in the rates of RII dephosphorylation in tissue extracts were not observed using purified RII from either tissue. These data suggested that the slow rate of RII dephosphorylation in rat hepatocytes is due to a difference in the susceptibility of endogenous rat liver RII to dephosphorylation rather than a difference in phosphatase activity.     

Autophosphorylation of cyclic AMP-dependent protein kinase from pig brain

Autophosphorylation of cyclic AMP-dependent pig brain protein kinase has been detected. Up to 1,5 moles of gamma-32P are transferred from [gamma-32P]ATP to the dimer of the regulatory subunit. The autophosphorylation reaction is Mg2+-dependent and occurs at a high rate: more than 50% of the radioactive label is incorporated during the first minute of incubation at 30 degrees. The pH dependence of this reaction differs from that of the phosphotransferase reaction. The phosphoholoenzyme is more sensitive to cyclic AMP than the dephosphoholoenzyme; however, both forms bind up to 2 moles of 3H-cyclic AMP per 1 mole of the holoenzyme. The activation and dissociation constants for both forms of the holoenzyme have been calculated. The autophosphorylation reaction has been shown to occur via an intramolecular mechanism; the phosphorylation of the regulatory subunit can occur only within the holoenzyme. The increase in the concentration of cyclic AMP causes the latter to produce an inhibitory effect on autophosphorylation. The regulatory action of autophosphorylation on cyclic AMP-dependent protein kinases is discussed.     

Autophosphorylation of the protein kinase dependent on double-stranded RNA

The double-stranded RNA (dsRNA)-dependent protein kinase (p68 kinase) from interferon-treated human cell is a Mr 68,000 protein induced by interferon. By the use of a specific monoclonal antibody, we have been able to study the two distinct protein kinase activities characteristic of purified p68 kinase. The first activity is functional for endogenous phosphorylation of the enzyme (p68 kinase), whereas the second one is responsible for the phosphorylation of exogenous substrates such as eukaryotic initiation factor 2 and histone. When activated by dsRNA in the presence of Mn2+ and ATP, p68 kinase is autophosphorylated and is then capable of catalyzing phosphorylation of histone in the absence of dsRNA. Whereas binding of 8-azido-[alpha-32P] ATP (8-N3ATP) to p68 kinase is dependent on both dsRNA and Mn2+, phosphorylated p68 kinase binds 8-N3ATP independent of dsRNA. This is consistent with a dsRNA requirement for the autophosphorylation of p68 kinase, but not for the phosphorylation of exogenous substrates. p68 kinase is mainly associated with the ribosomal pellet. It could be recovered efficiently by a buffer containing both high salt and a nonionic detergent. Synthesis of p68 kinase is induced several-fold by interferon in different types of human cells. Partial proteolysis of [35S]methionine and an 8-N3ATP-labeled p68 kinase preparation by Staphylococcus aureus V8 protease indicated the presence of a major Mr 48,000 polypeptide (p48) with a specific ATP-binding site. p48 probably contains the catalytic unit of p68 kinase and is analogous to a similar protein which we have previously described as a distinct protein present in a complexed form with p68 kinase. We now believe that the presence of p48 in previously purified kinase preparations was due to partial degradation of p68 kinase.     

Autophosphorylation of the catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase contains two stable phosphorylation sites, Thr-197 and Ser-338 (Shoji, S., Titani, K., Demaille, J. G., and Fischer, E. H. (1979) J. Biol. Chem. 254, 6211-6214). Thr-197 is very resistant to dephosphorylation and thus cannot typically be autophosphorylated in vitro once the stable subunit is formed. Ser-338 is slowly dephosphorylated and can be rephosphorylated autocatalytically. In addition to these two stable phosphorylation sites, a new site of autophosphorylation, Ser-10, was identified. Phosphorylation at Ser-10 does not have a major effect on activity, and phosphates from Ser-10 or Ser-338 are not transferred to physiological substrates such as the type II regulatory subunit. Autophosphorylation at Ser-10 is associated with one of the two major isoelectric variants of the catalytic subunit. The form having the more acidic pI can be autophosphorylated at Ser-10 while the more basic form of the catalytic subunit cannot. Phosphorylation at Ser-10 does not account for the two isoenzyme forms. Since the reason for two isoelectric variants of the catalytic subunit is still unknown, it is not possible to provide a structural basis for the difference in accessibility of Ser-10 to phosphorylation. Either Ser-10 is not accessible in the more basic form of the catalytic subunit or some other type of post- or cotranslational modification causes Ser-10 to be a poor substrate. Whether the myristoyl group at the amino-terminal Gly is important for Ser-10 autophosphorylation remains to be established. The isoenzyme forms of the catalytic subunit do not correspond to the gene products coded for by the C alpha and C beta genes.     

C-reactive protein suppresses insulin signaling in endothelial cells: role of spleen tyrosine kinase

Although few epidemiological studies have demonstrated that C-reactive protein (CRP) is related to insulin resistance, no study to date has examined the molecular mechanism. Here, we show that recombinant CRP attenuates insulin signaling through the regulation of spleen tyrosine kinase (Syk) on small G-protein RhoA, jun N-terminal kinase (JNK) MAPK, insulin receptor substrate-1 (IRS-1), and endothelial nitric oxide synthase in vascular endothelial cells. Recombinant CRP suppressed insulin-induced NO production, inhibited the phosphorylation of Akt and endothelial nitric oxide synthase, and stimulated the phosphorylation of IRS-1 at the Ser307 site in a dose-dependent manner. These events were blocked by treatment with an inhibitor of RhoA-dependent kinase Y27632, or an inhibitor of JNK SP600125, or the transfection of dominant negative RhoA cDNA. Next, anti-CD64 Fcgamma phagocytic receptor I (FcgammaRI), but not anti-CD16 (FcgammaRIIIa) or anti-CD32 (FcgammaRII) antibody, partially blocked the recombinant CRP-induced phosphorylation of JNK and IRS-1 and restored, to a certain extent, the insulin-stimulated phosphorylation of Akt. Furthermore, we identified that recombinant CRP modulates the phosphorylation of Syk tyrosine kinase in endothelial cells. Piceatannol, an inhibitor of Syk tyrosine kinase, or infection of Syk small interference RNA blocked the recombinant CRP-induced RhoA activity and the phosphorylation of JNK and IRS-1. In addition, piceatannol also restrained CRP-induced endothelin-1 production. We conclude that recombinant CRP induces endothelial insulin resistance and dysfunction, and propose a new mechanism by which recombinant CRP induces the phosphorylation of JNK and IRS-1 at the Ser307 site through a Syk tyrosine kinase and RhoA-activation signaling pathway.     

Structural insights into the autoactivation mechanism of p21-activated protein kinase

p21-activated kinases (PAKs) play an important role in diverse cellular processes. Full activation of PAKs requires autophosphorylation of a critical threonine/serine located in the activation loop of the kinase domain. Here we report crystal structures of the phosphorylated and unphosphorylated PAK1 kinase domain. The phosphorylated PAK1 kinase domain has a conformation typical of all active protein kinases. Interestingly, the structure of the unphosphorylated PAK1 kinase domain reveals an unusual dimeric arrangement expected in an authentic enzyme-substrate complex, in which the activation loop of the putative "substrate" is projected into the active site of the "enzyme." The enzyme is bound to AMP-PNP and has an active conformation, whereas the substrate is empty and adopts an inactive conformation. Thus, the structure of the asymmetric homodimer mimics a trans-autophosphorylation complex, and suggests that unphosphorylated PAK1 could dynamically adopt both the active and inactive conformations in solution.     

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.     

Differential in vitro phosphorylation of clathrin light chains by the epidermal growth factor receptor-associated protein tyrosine kinase and a pp60c-src-related spleen tyrosine kinase.

The epidermal growth factor (EGF) receptor-associated protein tyrosine kinase activity has been suggested to play important roles in the EGF-enhanced, clathrin-coated pit-mediated receptor internalization (W. S. Chen, C. S. Lazar, M. Peonie, R. Y. Tsien, G. N. Gill, and M. G. Rosenfeld, 1987, Nature 328, 820-823) but the kinase substrate important for this process has not been identified. This study demonstrates that the EGF receptor, partially purified from A431 epidermoid carcinoma cells, catalyzes the phosphorylation of one of the two clathrin light chains, clathrin light chain a (LCa). The phosphorylation activity is stimulated by EGF and immunoprecipitated by an EGF receptor monoclonal antibody. The phosphorylation occurs exclusively on tyrosine residues. Amino acid composition of the major tryptic phosphopeptide of the EGF receptor-phosphorylated LCa corresponds closely to that of residues 1 to 97 of LCa. A stoichiometry of 0.2 mol phosphate/mol LCa was attained after 60 min at 30 degrees C and a Km value of 1.7 microM was determined for the reaction. LCa of either neuronal or non-neuronal origin could serve as a substrate. In addition to the EGF receptor tyrosine kinase, a particulate src-related protein tyrosine kinase purified from bovine spleen (C. M. E. Litwin, H.-C. Cheng, and J. H. Wang, 1991, J. Biol. Chem. 226, 2557-2566) was shown in this study to also phosphorylate the light chains. However, in contrast to the EGF receptor phosphorylation, both clathrin light chains a and b were phosphorylated by the spleen kinase, suggesting that the two tyrosine kinases have differential site specificities. Given the specificity of LCa phosphorylation by the EGF receptor, we propose that LCa phosphorylation on a tyrosine residue(s) may be important in EGF-induced receptor internalization.     

Autophosphorylation and rapid dephosphorylation of the cAMP-dependent protein kinase from Blastocladiella emersonii zoospores

The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate and affinity chromatography on N6-(2-aminoethyl)-cAMP-Sepharose were used to analyze the cAMP-binding proteins present in cell-free extracts of Blastocladiella emersonii zoospores. In the presence of a mixture of protease inhibitors, 8-azido[32P]cAMP was specifically and quantitatively incorporated into a major protein band of Mr = 58,000, and three minor protein bands of Mr = 50,000, Mr = 43,000, and Mr = 36,000 respectively, after autoradiography following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. In the absence of the protease inhibitors, the Mr = 58,000 protein band was converted into the lower molecular weight cAMP-binding proteins, indicating a high sensitivity of the intact Mr = 58,000 protein band to endogenous proteases. The Mr = 58,000 protein corresponded to the regulatory subunit (R), of the cAMP-dependent protein kinase of zoospores, as shown by their identical behavior on DEAE-cellulose chromatography. The partially purified protein kinase incorporated 32P from [gamma-32P] ATP . Mg2+ into R as demonstrated by the specific adsorption of the 32P-labeled protein with N6-(2-aminoethyl)-cAMP-Sepharose. The incorporated 32P group was rapidly removed by endogenous phosphoprotein phosphatases in the presence of cAMP, as shown by pulse-chase experiments with [gamma-32P]ATP. Dephosphorylation of R-cAMP and rapid proteolysis may indicate two other mechanisms, in addition to cAMP, for the control of this protein kinase in vivo.     

cGMP-dependent protein kinase. Autophosphorylation changes the characteristics of binding site 1

cGMP-dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg . ATP, whereas binding to site 2 (apparent Kd 100-150 nM) is non-cooperative and not affected by Mg . ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg . ATP. The association (K1) and dissociation (K-1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg . ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg . ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500-fold dilution of the enzyme-ligand complex is 100-fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg . ATP or autophosphorylation. Only Kd values calculated with the latter K-1 values are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP-dependent protein kinase affects mainly the binding characteristics of site 1.