Cyclic AMP-dependent and -independent protein kinases of the water mold, Blastocladiella emersonii.

Protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) and cyclic adenosine 3',5'-monophosphate binding activities have been identified in zoospore extracts of the water mold Blastocladiella emersonii. More than 75% of these activities is found in the soluble fraction. Soluble protein kinase activity is resolved in three peaks(I, II and III) by DEAE-cellulose chromatography. Peak I is casein dependent and insensitive to cyclic AMP. Peak II is histone dependent and cyclic AMP independent; this enzyme is inhibited by the heat-stable inhibitor from bovine muscle. Peak III utilizes histone as substrate and is activated by cyclic AMP.     

Phosphorylation of fibrinogen by casein kinase 2.

Casein kinase 2 from rat liver cytosol phosphorylated human fibrinogen in a reaction that was not stimulated by Ca2+ or cyclic AMP, but was markedly inhibited by heparin, and proceeded at a similar rate when either ATP or GTP was used as phosphate donor. Analysis of casein kinase 2 by glycerol-density-gradient centrifugation showed that the activities towards fibrinogen, casein, phosvitin, high-mobility-group protein 14 and glycogen synthase coincided. Maximal incorporation into fibrinogen by casein kinase 2 averaged 1 mol of phosphate/mol of protein substrate, most of it in the alpha-chain, although some phosphorylation of the beta-chain was also detected. Analysis of phosphorylated alpha-chain revealed that most of the phosphate was incorporated on serine. Phosphorylation of human fibrinogen was also performed by casein kinase 2 from human polymorphonuclear leucocytes, lymphocytes and platelets.     

Purification and characterization of a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands. Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 300 mM, phosphorylated only phosvitin and was not retained on phosphocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhibited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent Km of 100 micrograms/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent Km of 25 and 28 microM, respectively. It had an absolute requirement for Mg2+ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecular weight of 35000 suggesting a polymeric structure of the enzyme.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.     

An analysis of the autophosphorylation of rabbit and human erythrocyte membranes.

The autophosphorylation of rabbit and human erythrocyte membranes has been studied under various experimental conditions. The phosphopeptides of the erythocyte membranes were identified using sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by ratioautography. The pattern of phosphorylatiion of membrane components differs with respect to the phosphoryl donor used (ATP or GTP) and to the pH at which the reaction is carried out. Both species appear to contain at least two distinct membrane-bound protein kinases. The human erythrocyte membrane contains a cyclic adenosine 3'5'-monophosphate (cyclic AMP)-dependent protein kinase and several substrates for this kinase. Only ATP can be used as a phosphoryl donor for this kinase. In contrast, the rabbit erythrocyte membrane does not contain a cyclic AMP dependent protein kinase but does contain a kinase which utilizes only ATP as the phosphoryl donor and is specific for certain endogenous substrates at low pH. Both the human and rabbit erythrocyte membranes contain a kinase which utilizes GTP, perhaps also ATP, as the phosphoryl donor. The substrates of these kinases are similar in both species.     

Effects of isoproterenol on cyclic-AMP metabolism in rat ventral prostate.

Beta-Adrenergic stimulation of the ventral prostate cyclic-AMP system was investigated by examining the influence of isoproterenol on endogenous cyclic-AMP levels as well as on the activities of adenylate cyclase CEC 4.6.1.1) and cyclic-AMP-dependent and independent protein kinases (EC 2.7.1.37). Administration of isoproterenol (1 mg/kg, ip) resulted in rapid elevation of adenylate cyclase activity (119%) and cyclic-AMP levels (593%). The observed isoproterenol-stimulated changes in cyclic-AMP metabolism of the ventral prostate were time-dependent and maximal stimulation was seen 5 min after treatment with this beta-adrenergic agonist. The increases in prostatic adenylate cyclase and cyclic-AMP also were related to the dose of isoproterenol administered and maximal enhancement of these parameters was seen with 1 mg/kg dose of the agonist. Whereas pretreatment of rats with propranolol (3mg/kg, ip) partially reversed these alterations, administration of an alpha-adrenergic antagonist, phentolamine, even at a dose of 5 mg/kg, failed to elicit any appreciable effect. Stimulation of prostatic soluble protein kinase by isoproterenol was associated with a decrease (33%) in the activity of the cyclic-AMP-dependent protein kinase with a concomitant increase (25%) in that of the independent enzyme. Whereas the ability of the enzyme to bind cyclic-(3H) AMP in vitro was decreased (54%) following isoproterenol treatment, the protein kinase activity ratio (-cyclic-AMP/+cyclic AMP) was significantly elevated from 0.51+/0.05 to 0.95+/0.08. Although propranolol alone had little or no effect on these parameters, it inhibited partially the isoproterenol-induced alterations in cyclic-AMP-dependent protein kinase and the cyclic-AMP binding capacity. Treatment with propranolol also blocked the increases in the kinase activity ratio and in the activity of cyclic-AMP-independent enzyme seen with isoproterenol. Data suggest that the concentration of ventral prostate cyclic-AMP as well as the activities of adenylate cyclase and cyclic-AMP-dependent and independent form of protein kinases are subject to modulation by beta-adrenergic stimulation.     

GTP,as well as ATP,can act as a substrate for the intrinsic protein kinase activity of synaptic plasma membranes

Summary GTP as well as ATP can act as phosphate donor for the intrinsic protein kinase activity of synaptic plasma membranes. There are many similarities between the activities observed with ATP or GTP. Both need a divalent cation, Mg²⁺ being preferred, both are slightly inhibited by Na⁺, and more strongly by K⁺, both are inhibited by theophylline and adenosine. The K_m for GTP (0.13 mM) is similar to that ATP (0.12 mM). There are, however, some differences in properties. When GTP instead of ATP is the phosphate donor the pH optimum is 6.5 instead of 7.4. In addition NH ₄ ⁺ inhibits the transfer of phosphate from GTP but not from ATP. More importantly, cyclic AMP only stimulates the transfer of phosphate from ATP not from GTP. SDS gel electrophoresis reveals that similar membrane proteins are phosphorylated by GTP and ATP in the presence or absence of cyclic AMP. This suggests that there may be two different types of protein kinase in the synaptic plasma membrane which act on similar membrane proteins. One is stimulated by cyclic AMP and is specific to ATP while the other is unaffected by cyclic nucleotides and can use either ATP or GTP as phosphate donor.Deceased     

Fractionation of two protein kinases from avian myeloblastosis virus and characterization of the protein kinase activity preferring basic phosphoacceptor proteins.

Two protein kinase activities were fractionated from purified virions of avian myeloblastosis virus. Distinguishing characteristics of these two protein kinases included: (i) their binding properties during purification by ion-exchange chromatography; (ii) their estimated molecular weights; and (iii) their phosphoacceptor protein specificities. The protein kinase that bound to the anion exchanger DEAE-cellulose (pH 7.2) had an estimated molecular weight of 60,000 to 64,000 and preferred basic phosphoacceptor proteins. The protein kinase that bound to the cation exchanger phosphocellulose (pH 7.2) had an estimated molecular weight of 42,000 to 46,000 and preferred acidic phosphoacceptor proteins. The protein kinase preferring basic phosphoacceptor proteins was further purified and characterized. Optimal transfer of phosphate catalyzed by this enzyme required a divalent metal ion, a sulfhydryl-reducing agent, and ATP as phosphate donor. GTP was not an effective phosphate donor at concentrations comparable to ATP; and the cyclic nucleotides cyclic AMP and cyclic GMP neither stimulated nor inhibited protein phosphorylation by the protein kinase. The specificity of the protein kinase for basic phosphoacceptor proteins extended to proteins from avian myeloblastosis virus, in that the neutral to basic virion proteins p12, p19, and p27 served as phosphate acceptors. In addition, the protein kinase also appeared to phosphorylate itself. The role(s) of this virion-associated protein kinase is discussed.     

Protein kinase activities in rat pancreatic islets of Langerhans.

1. Protein kinase activities in homogenates of rat islets of Langerhans were studied. 2. On incubation of homogenates with [gamma-32P]ATP, incorporation of 32P into protein occurred: this phosphorylation was neither increased by cyclic AMP nor decreased by the cyclic AMP-dependent protein kinase inhibitor described by Ashby & Walsh [(1972) J. Biol. Chem. 247, 6637--6642]. 3. On incubation of homogenates with [gamma-32P]ATP and histone as exogenous substrate for phosphorylation, incorporation of 32P into protein was stimulated by cyclic AMP (approx. 2.5-fold) and was inhibited by the cyclic AMP-dependent protein kinase inhibitor. In contrast, when casein was used as exogenous substrate, incorporation of 32P into protein was not stimulated by cyclic AMP, nor was it inhibited by the cyclic AMP-dependent protein kinase inhibitor. 4. DEAE-cellulose ion-exchange chromatography resolved four peaks of protein kinase activity. One species was the free catalytic subunit of cyclic AMP-dependent protein kinase, two species corresponded to 'Type I' and 'Type II' cyclic AMP-dependent protein kinase holoenzymes [see Corbin, Keely & Park (1975) J. Biol. Chem. 250, 218--225], and the fourth species was a cyclic AMP-independent protein kinase. 5. Determination of physical and kinetic properties of the protein kinases showed that the properties of the cyclic AMP-dependent activities were similar to those described in other tissues and were clearly distinct from those of the cyclic AMP-independent protein kinase. 6. The cyclic AMP-independent protein kinase had an s20.w of 5.2S, phosphorylated a serine residue(s) in casein and was not inhibited by the cyclic AMP-dependent protein kinase inhibitor. 7. These studies demonstrate the existence in rat islets of Langerhans of multiple forms of cyclic AMP-dependent protein kinase and also the presence of a cyclic AMP-independent protein kinase distinct from the free catalytic subunit of cyclic AMP-dependent protein kinase. The presence of the cyclic AMP-independent protein kinase may account for the observed characteristics of 32P incorporation into endogenous protein in homogenates of rat islets.     

Changes in cyclic AMP-dependent protein dinase activity in Tetrahymena pyriformis during the growth cycle.

An adenosine 3':5'-monophosphate-dependent protein kinase II (ATP:protein phosphotransferase, EC 2.7.1.37) was partially purified from the cytosol fraction of an exponentially growing culture of Tetrahymena pyriformis. Protein kinase II represented approximately 90% of the cytosolic protein kinase activity. The enzyme had a high degree of substrate specificity for calf thymus and Tetrahymena histones as compared to casein, protamine and phosvitin. The enzyme incorporated the terminal phosphate of ATP into serine and threonine residues of all the histone fractions. The apparent Km of the enzyme for adenosine 3':5'-monophosphate (cyclic AMP) was 1-10-minus 8 M. Protein kinase II was also activated by other cyclic nucleotides with apparent Km values in the range 2.k-10-minus 6 M. Ther specific activity of the cyclic AMP-dependent protein kinase of Tetrahymena decreases markedly from initial high values during the transition from the lag to early log phase of growth. This is followed by a shrp increase in the activity of the enzyme as the log phase of growth progresses. The specific activity of the enzyme increases rapidly during the heat-induced synchronization of Tetrahymena cells. The capacity for rapid phosphorylation of multiple classed of organelle-specific phosphoproteins and the level of cyclic AMP were maximal in Tetrahymena during the earliest phase of growth. These results demonstrate that the cell cycle of Tetrahymena may be coordinated by marked variations in the level of cyclic AMP which in turn regulate the cyclic AMP-dependent protein kinase.     

Identification of a novel casein kinase activity in HeLa cell nuclei

Three casein kinase activities have been resolved by column chromatography of HeLa cell nuclear extracts. In addition to casein kinases NI and NII, which have been described in other cell types, HeLa nuclei contain a third casein kinase activity which we have named NIII. NIII is a cyclic nucleotide-independent casein kinase which uses either Mg2+ or Mn2+ as a divalent cation, but is inhibited by increasing NaCl concentrations in the presence of Mg2+ and has optimal activity at 50 mM NaCl in the presence of Mn2+. In Mg2+, NIII uses only ATP as a phosphate donor, but in Mn2+ NIII transfers phosphate from either ATP or GTP. NIII phosphorylates the serine and threonine residues of casein, but does not phosphorylate phosvitin or calf thymus histones.     

Purification and characterization of two casein kinases from ejaculated bovine spermatozoa.

Two protein kinases active on casein and phosvitin were partially purified from the soluble fraction of ejaculated bovine spermatozoa. They were operationally termed casein kinase A and B based on the order of their elution from a phosphocellulose column. CK-A showed an approximate molecular mass of 38 kDa, and it phosphorylated serine residues of casein and phosvitin utilizing ATP as a phosphate donor (Km 19 microM). Enzyme activity was maximal in the presence of 10 mM MgCl2, whereas it decreased in the presence of spermine, polylysine, quercetin, and NaCl (20-250 mM). CK-B seemed to have a monomeric structure of about 41 kDa; it underwent autophosphorylation and cross-reacted with polyclonal antibodies raised against recombinant alpha, but not beta, subunit of human type 2 casein kinase. It phosphorylated both serine and threonine residues of casein and phosvitin, utilizing ATP (Km 12 microM) but not GTP as a phosphate donor. Threonine was more affected in the phosphorylated phosvitin than in the partially dephosphorylated substrate. CK-B was active toward the synthetic peptide Ser-(Glu)5 and calmodulin (in the latter case, in the presence of polylysine), and it was activated by spermine, polylysine, MgCl2 (30 mM), and NaCl (20-400 mM). The activity of the enzymes was not affected by cAMP, or the heat-stable inhibitor of the cAMP-dependent protein kinase, or calcium.     

Properties of neurofilament protein kinase.

Neurofilament (NF) protein kinase, partially purified from NF preparations [Toru-Delbauffe & Pierre (1983) FEBS Lett. 162, 230-234], was found to be distinct from both the casein kinase present in NFs and the cyclic AMP-dependent protein kinase which is able to phosphorylate NFs. NF-kinase phosphorylated the three NF protein components. The amount of phosphate incorporated per molecule was higher for NF 200 than for NF 145 and NF 68. Other proteins present in the NF preparations were also used as NF-kinase substrates. Two of them might correspond to the myelin basic proteins with Mr values of 18,000 and 21,000. Four other substrates in the NF preparation were not identified (respective Mr values 53,000, 55,000, 65,000 and greater than 300,000). NF kinase also phosphorylated two additional brain-cell cytoskeletal elements: GFAp and vimentin. Casein, histones and phosvitin, currently used as substrates for protein kinase assays, were very poor phosphate acceptors. Half-maximal NF-kinase activity was obtained at an NF protein concentration of about 0.25 mg/ml in heated, salt-washed, NF preparations. The specific activity was about 5 pmol of 32P incorporated/min per microgram of NF kinase preparation protein. ATP was a phospho-group donor (Km 8 X 10(-5) M), but GTP was not. NF-kinase activity remained stable at 65 degrees C for more than 1 h. The enzyme was not degraded by storage at -20 degrees C for several months in a buffer containing 50% (w/v) sucrose. Maximal activity was obtained with 5 mM-Mg2+ (Mg2+ could be replaced by Co2+); Zn2+ and Cu2+ inhibited the reaction. NF-kinase was not dependent on cyclic AMP, cyclic GMP, Ca2+ or Ca2+ plus dioleoylglycerol and phosphatidylserine.     

Purification and properties of a virion protein kinase.

The protein kinase associated with virions of frog virus 3 was purified to apparent homogeneity by ion exchange chromatography and gel filtration. The enzyme protein appeared as a single polypeptide of molecular weight 50,000 to 55,000 as determined by gel filtration, glycerol gradient sedimentation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and comprised approximately 0.4% of the total virion protein. The activity was classified as a cyclic nucleotide-independent protein kinase as it was not effected by cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate, or inhibited by a cyclic nucleotide-dependent protein kinase inhibitor protein, and utilized GTP as well as ATP as a phosphate donor. The greatest rates of phosphorylation were obtained with acidic phosphoprotein substrates such as casein or phosvitin, although potential physiological substrates for this activity included specific virion polypeptides of frog virus.     

Multiple protein kinases from human lymphocytes. Identification enzymes phosphorylating exogenous histon and casein.

1. Cell-free lysates of human peripheral blood lymphocytes contained two casein kinase activities and two histone kinase activities, which could be separated by chromatography on DEAE-Sephadex. 2. Neither of the casein kinase activities were stimulated by cyclic AMP. The major activity was eluted from DEAE-Sephadex between 0.4 and 0.45M-KCl, had a molecular weight of approx. 130,000 (sucrose density gradients) and was stimulated by KCl (maximum 150mM). It also formed higher-molecular-weight aggregates when centrifuged in sucrose gradients containing 150mM-KCl. The minor activity was not retained by DEAE-Sephadex, had a molecular weight of approx. 50,000 and was not stimulated by KCl. 3. The major histone kinase activity was stimulated by cyclic AMP and was eluted from the DEAE-Sephadex column between 0.05 and 0.2M-KCl. The other activity was not stimulated by cyclic AMP and was insensitive to the rabbit muscle protein kinase inhibitor. 4. Evidence was obtained suggesting that the lymphocyte casein kinases were located primarily in the nuclei.     

Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [³H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [³H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors.Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [³H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [³H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [³H]AMP for renal receptors. The cyclic [³H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [³H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [³H]AMP was enhanced by ATP or AMP.Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N⁶-butyryl-adenosine cyclic 3′,5′-monophosphate or N⁶,O²-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [³H]AMP in competitive binding assays.This study suggested that the membrane cyclic [³H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.     

Identification and solubilization of a cAMP-independent protein kinase from mouse L-cell smooth membranes

1. Smooth membranes have been prepared from mouse L-cells and found to contain an endogenous protein kinase activity. 2. The enzyme(s) responsible for this activity use ATP, but no other nucleoside triphosphates, to phosphorylate endogenous membrane proteins as well as exogenously-added protein substrates such as phosvitin and casein. 3. Mg2+ is required for enzyme activity, maximal activity is observed at pH 7.5-8.0 and the kinase is not dependent on, or stimulated by, cyclic 3'-5' AMP. 4. The kinase activity is not decreased by the Walsh heat-stable inhibitor of cyclic 3'-5' AMP-dependent protein kinases. 5. Fifty percent or more of the membrane-associated kinase activity can be solubilized by extracting membranes with buffer containing 0.6 M NaCl. 6. The solubilized enzyme resembles the membrane-associated activity in its Mg2+ requirement, pH optimum and independence of cyclic 3'-5' AMP. 7. Phosvitin and casein are better exogenous substrates than histones or protamine for phosphorylation by the enzyme in either the membrane-associated or solubilized state.     

Cyclic AMP-dependent protein kinase of Neurospora crassa

$\text{Neurospora}\text{crassa}$ was surveyed for cyclic AMP-dependent protein kinase activity. Two peaks (I and II) of protein kinase activity were demonstrated by DEAE-cellulose chromatography of wild type $\text{Neurospora}$ extracts. Peak I was stimulated by cyclic AMP, eluted below 60 mM NaCl and had high activity using histone H2B as substrate. Peak II eluted at 200–250 mM NaCl; its activity was not cyclic AMP stimulated and was highest with dephosphorylated casein as a substrate. Cyclic AMP binding to a protein associated with the protein kinase is specifically inhibited by certain cyclic AMP analogs.     

Phosphorylation of fibrinogen by casein kinase 1

Casein kinase 1 phosphorylated human fibrinogen, in a reaction that did not use GTP as phosphoryl donor and was neither stimulated by cyclic AMP or Ca2+, nor inhibited by the cyclic AMP-dependent protein kinase inhibitor protein. Maximal incorporation averaged 4 mol of phosphate per mol of fibrinogen, most of it in the largest CNBr-fragment of the alpha-chain. Phosphoamino acid analysis revealed that phosphorylation occurred only at seryl residues. The phosphorylation of fibrinogen by casein kinase 1 was reverted by alkaline phosphatase.     

A Protein Kinase Activity Is Associated with and Specifically Phosphorylates the Neural Cell Adhesion Molecule LI

The neural cell adhesion molecule L1 is a phosphorylated integral membrane glycoprotein that is recovered from adult mouse brain by immunoaffinity chromatography as a set of polypeptides with apparent molecular masses of 200, 180, 140, 80, and 50 kilodaltons (L1-200, L1-180, L1-140, L1-80, and L1-50, respectively). In the present study, we show that two kinase activities are associated with immunopurified L1: One specifically phosphorylates L1-200 and L1-80 but not L1-180, L1-140, or L1-50. This pattern of phosphorylation corresponds to the one described for L1 after metabolic phosphate incorporation into cultures of cerebellar cells. In both cases, serine is the main amino acid that is labeled by radioactive phosphate. The kinase activity is not activated by Ca2+, calmodulin, phosphatidylserine, diolein, cyclic AMP, or cyclic GMP, a result suggesting that the enzyme is distinct from Ca2+/calmodulin-dependent kinases, from protein kinase C, or from cyclic AMP/cyclic GMP-dependent kinases and may belong to the independent kinase group. The other kinase phosphorylates only casein but not L1, utilizes GTP as well as ATP, and is strongly inhibited by heparin. Because the primary structure of the L1 protein does not contain consensus sequences characteristic for known kinases, we believe that the catalytic activities detectable in immunopurified L1 are due to kinases that are strongly enough associated with L1 to withstand the stringent purification procedures.