Phosphate Dependent Protein Kinase Research Articles

Research Progress of PI3K/PTEN/AKT Signaling Pathway Associated with Renal Cell Carcinoma.

Renal cell carcinoma is a common renal malignancy of the urinary system and the most malignant type of kidney cancer. Phosphatidylinositol 3-kinase (PI3K) is an intracellular phosphatidylinositol kinase associated with oncogene products such as v-src and with serine/threonine kinase activity, and its increased activity correlates with the development of several cancers. Protein kinase B (AKT) is a cyclic guanosine phosphate-dependent protein kinase that plays an important role in cell survival and apoptosis. Phosphatase and tensin homolog (PTEN), a newly discovered oncogene in recent years, participates in tumorigenesis and development by competing with tyrosine kinases for common substrates. The product encoded by PTEN was found to negatively regulate the PI3K/Akt signaling pathway, thereby inhibiting cell proliferation and promoting apoptosis. The PI3K/PTEN/AKT signaling pathway has also been identified in several studies as being involved in the development of several malignancies, including renal cell carcinoma. Radiotherapy is currently one of the most effective means of treatment for renal cell carcinoma, whereas it is predisposed to significant tolerance during the course of radiotherapy, thereby leading to treatment failure. Therefore, new treatment options may potentiate the efficiency of renal cell carcinoma treatment. With the development of tumor molecular biology, targeted biological therapy for malignant tumors has gradually become a research hotspot. Given the above research background, this study reviews the application of the PI3K/PTEN/AKT signaling pathway in renal cell carcinoma, aiming to provide more references for the treatment of clinical renal cell carcinoma.

Heregulin-dependent activation of phosphoinositide 3-kinase and Akt via the ErbB2/ErbB3 co-receptor.

The ErbB2/ErbB3 heregulin co-receptor has been shown to couple to phosphoinositide (PI) 3-kinase in a heregulin-dependent manner. The recruitment and activation of PI 3-kinase by this co-receptor is presumed to occur via its interaction with phosphorylated Tyr-Xaa-Xaa-Met (YXXM) motifs occurring in the ErbB3 C terminus. In this study, mutant ErbB3 receptor proteins expressed in COS7 cells were used to investigate PI 3-kinase-dependent signaling pathways activated by the ErbB2/ErbB3 co-receptor. We observed that a mutant ErbB3 protein with each of its six YXXM motifs containing a Tyr --> Phe substitution was unable to bind either the p85 regulatory or p110 catalytic subunit of PI 3-kinase. However, restoration of a single YXXM motif was sufficient to mediate association with the PI 3-kinase holoenzyme, although at a lower level than wild-type ErbB3. When ErbB3 YXXM motifs were restored in pairs, evidence for cooperativity between two, those incorporating Tyr-1273 and Tyr-1286, was observed. Interestingly, we have shown that an apparent association of PI 3-kinase activity with ErbB2/Neu was due to the residual presence of ErbB3 in ErbB2 immunoprecipitates. The necessity of ErbB3 association with PI 3-kinase for downstream signaling to the effector kinase Akt was also investigated. Here, the heregulin-dependent translocation of Akt to the plasma membrane and its subsequent activation was observed in intact NIH-3T3 fibroblasts. Recruitment of PI 3-kinase to ErbB3 was required for both activities, and it appeared that ErbB2 activation alone was not sufficient to activate PI 3-kinase signaling in these cells.

Binding of 9-Anthroylcholine Monitors the Interactions of Adenosine Cyclic 3′,5′-Phosphate-dependent Protein Kinase with MgATP, Substrates, and Regulatory Subunits

The isolated catalytic subunit of cAMP-dependent protein kinase and smooth muscle myosin light chain kinase undergo interactions with the fluorescent dye 9-anthroylcholine (9AC) that are responsive to the two enzymes' associations with substrates and effectors. Additionally, the binding of 9AC is highly sensitive to subtle structural or functional differences among closely related protein kinases. Skeletal muscle myosin light chain kinase and the catalytically active chymotryptic fragment of the gamma-subunit of phosphorylase kinase do not associate with 9AC. The 1:1 fluorescent complex of the isolated catalytic subunit of cAMP-dependent protein kinase with 9AC exhibits a dissociation constant of 21 microM. The association of the catalytic subunit with either of the regulatory subunits, RI and RII, results in decreases in the observed 9AC fluorescence that are reversed upon the addition of cAMP. The effects of MgATP and of polypeptide substrates (Kemptide, troponin I, protamine) on the 9AC-catalytic subunit complex are consistent with a general noncompetitive model in which the interactions of 9AC and the other ligands with the enzyme are mutually antagonistic but not purely competitive.

Identification of phosphorylation sites for adenosine 3',5'-cyclic phosphate dependent protein kinase on the voltage-sensitive sodium channel from Electrophorus electricus

The voltage-sensitive sodium channel from the electroplax of Electrophorus electricus is selectively phosphorylated by the catalytic subunit of cyclic-AMP-dependent protein kinase (protein kinase A) but not by protein kinase C. Under identical limiting conditions, the protein was phosphorylated 20% as rapidly as the synthetic model substrate kemptamide. A maximum of 1.7 +/- 0.6 equiv of phosphate is incorporated per mole. Phosphoamino acid analysis revealed labeled phosphoserine and phosphothreonine at a constant ratio of 3.3:1. Seven distinct phosphopeptides were identified among tryptic fragments prepared from radiolabeled, affinity-purified protein and resolved by HPLC. The three most rapidly labeled fragments were further purified and sequenced. Four phosphorylated amino acids were identified deriving from three consensus phosphorylation sites. These were serine 6, serine 7, and threonine 17 from the amino terminus and a residue within 47 amino acids of the carboxyl terminus, apparently serine 1776. The alpha-subunits of brain sodium channels, like the electroplax protein, are readily phosphorylated by protein kinase A. However, these are also phosphorylated by protein kinase C and exhibit a markedly different pattern of incorporation. Each of three brain alpha-subunits displays an approximately 200 amino acid segment between homologous repeat domains I and II, which is missing from the electroplax and skeletal muscle proteins [Noda et al. (1986) Nature (London) 320, 188; Kayano et al. (1988) FEBS Lett. 228, 1878; Trimmer et al. (1989) Neuron 3, 33]. Most of the phosphorylation of the brain proteins occurs on a cluster of consensus phosphorylation sites located in this segment. This contrasts with the pattern of highly active sites on the amino and carboxyl termini of the electroplax protein. The detection of seven labeled tryptic phosphopeptides compared to the maximal labeling stoichiometry of approximately 2 suggests that many of the acceptor sites on the protein may be blocked by endogenous phosphorylation.

Role of the Cyclic AMP—Dependent Protein Kinase in Signal Transduction

The first example of an enzyme that undergoes phosphorylation and dephosphorylation was phosphorylase, which catalyzes the initial step in the breakdown of tissue glycogen, a process that occurs when cells are stimulated by certain hormones. The phosphorylation reaction that activates is catalyzed by an enzyme designated phosphorylase whereas the dephosphorylation reaction is catalyzed by an enzyme called phosphorylase phosphatase. Phosphorylase kinase, like itself, is regulated by phosphorylation-dephosphorylation. In this instance the enzyme is phosphorylated and activated by a protein kinase that is the intracellular target of adenosine 3′,5′-cyclic phosphate, the second messenger of hormone action that was discovered by the late Earl Sutherland. Knowledge of these steps led to the establishment of the glycogenolytic cascade in which adenosine 3′,5′-cyclic phosphate, formed as a result of hormone action, first activates the adenosine 3′,5′-cyclic phosphate—dependent protein kinase, which in turn activates kinase, which then converts inactive to its active form. This is followed by the breakdown of glycogen. ( JAMA . 1989;262:1815-1818)

The Albert Lasker Medical Awards. Role of the cyclic AMP-dependent protein kinase in signal transduction

The first example of an enzyme that undergoes phosphorylation and dephosphorylation was phosphorylase, which catalyzes the initial step in the breakdown of tissue glycogen, a process that occurs when cells are stimulated by certain hormones. The phosphorylation reaction that activates phosphorylase is catalyzed by an enzyme designated "phosphorylase kinase," whereas the dephosphorylation reaction is catalyzed by an enzyme called "phosphorylase phosphatase." Phosphorylase kinase, like phosphorylase itself, is regulated by phosphorylation-dephosphorylation. In this instance the enzyme is phosphorylated and activated by a protein kinase that is the intracellular target of adenosine 3',5'-cyclic phosphate, the second messenger of hormone action that was discovered by the late Earl Sutherland. Knowledge of these steps led to the establishment of the glycogenolytic cascade in which adenosine 3',5'-cyclic phosphate, formed as a result of hormone action, first activates the adenosine 3',5'-cyclic phosphate-dependent protein kinase, which in turn activates phosphorylase kinase, which then converts inactive phosphorylase to its active form. This is followed by the breakdown of glycogen.

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.

Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide.

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase (EC 2.7.1.3) in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent Ki of 60 microM. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD included (1) modification with [14C]DCCD, (2) modification by DCCD in the presence of [3H]aniline, and (3) modification with DCCD and [14C]glycine ethyl ester. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformation of Leu-Arg-Arg-Ala-Ser-Leu-Gly bound in the active site of adenosine cyclic 3',5'-phosphate dependent protein kinase.

Studies utilizing NMR spectroscopy have shown that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) probably binds Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) in one of two extended coil conformations (A or B). The relative reactivities of a series of N-methylated peptides based on the structure of peptide 1 might, therefore, be related to how well each can assume the A or B conformation. From estimates of the magnitude of steric interactions that would be induced by N-methylation of an amide in peptide 1 that is locked in either conformation, the ability of each peptide to form that conformation was predicted. The ability of A-kinase to catalyze phosphorylation of the N-methylated peptides correlated well with the ability of each peptide to form conformation A, but not conformation B. In accord with these findings, the reactivity of an unreactive N-methylated peptide was partially restored by a second change, which allowed the peptide to assume conformation A. These results suggest that, when bound in the enzymatic active site, peptide 1 has a conformation that resembles structure A much more closely than structure B.

Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities.

As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3',5'-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-Kinase catalyzes the phosphorylation of five N alpha-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N alpha-methylated and depsi-containing peptide 1 analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

Smooth muscle myosin light chain kinase. Amino acid sequence at the site phosphorylated by adenosine cyclic 3',5'-phosphate-dependent protein kinase whether or not calmodulin is bound.

Turkey gizzard smooth muscle myosin light chain kinase is a calmodulin-dependent enzyme containing 2 serine residues that can be phosphorylated by cAMP-dependent protein kinase. One of these sites can be phosphorylated only when calmodulin is not bound to the enzyme; the amino acid sequence around this site has been reported recently (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464). Here we report the sequence around the site that is phosphorylated by cAMP-dependent protein kinase whether or not calmodulin is bound: Lys-Ala-Ser(P)-Gly-Ser-Ser-Pro-Thr-Ser-Pro-Ile-Asn-Ala-Asp-Lys-Val-Glu-A sn-Glu- . This sequence conforms to the previously defined criteria for substrates of cAMP-dependent protein kinase.

Association of calmodulin with peptide analogues of the inhibitory region of the heat-stable protein inhibitor of adenosine cyclic 3',5'-phosphate dependent protein kinase.

A 20-residue peptide analogue (IASGRTGRRNAIHDILVSSA) of the 8000-dalton heat-stable cAMP-dependent protein kinase inhibitor undergoes efficient calcium-dependent binding by calmodulin, with Kd approximately 70 nM when calcium is present. It is a potent inhibitor of smooth muscle myosin light chain kinase and of the calmodulin-dependent phosphatase activity of calcineurin. At concentrations above 3 microM, the peptide stimulates the basal activity of calcineurin. The native protein kinase inhibitor has no effect on the catalytic activity of myosin light chain kinase and is moderately inhibitory to both the calmodulin-dependent and -independent phosphatase activity of calcineurin. Competition experiments using excess concentrations of calcineurin and calmodulin suggest that the primary interaction of the native heat-stable inhibitor is with the catalytic subunit of protein kinase. Dansylcalmodulin exhibits only a weak interaction with the inhibitor. Observations on deletion peptides of the 20-residue analogue help to delineate the overlapping peptide binding specificities of the cAMP-dependent protein kinase [Scott, J. D., Glaccum, M. B., Fischer, E. H., & Krebs, E. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1613-1616] and calmodulin. In both cases, the most effectively bound peptides contain the RTGRR sequence.

Calmodulin-linked equilibria in smooth muscle myosin light chain kinase.

Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., & Shalitin, Y. S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5'-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 microM each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 microM enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 +/- 0.08 microM calcium. Three proteolytic fragments of smooth muscle myosin light chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96K fragment to approximately 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25 degrees C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3',5'-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., & Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase phosphotransferase activity. The results show that smooth muscle myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.

Conversion of bovine cardiac adenosine cyclic 3',5'-phosphate dependent protein kinase to a heterodimer by removal of 45 residues at the N-terminus of the regulatory subunit.

The type II adenosine cyclic 3',5'-phosphate (cAMP) dependent protein kinase from bovine heart, consisting of a dimeric regulatory subunit and two catalytic subunits, was converted to a heterodimer by limited tryptic digestion. Loss of the tetrameric structure was accompanied by proteolysis of the regulatory subunit to a form with an apparent molecular weight of 45 000 vs. 52 000 for the native subunit. The proteolyzed subunit behaved as a monomer, in contrast to the dimeric native subunit. Amino acid sequence analysis established that proteolysis removed 45 residues at the N-terminus, indicating that these 45 residues constitute the dimerizing domain of this protein. The kinetic properties of this heterodimer were indistinguishable from those of the native tetramer: half-maximal kinase activation occurred at 48 nM cAMP with a Hill coefficient of 1.45, the regulatory subunit bound 1.5 equiv of cAMP with half-maximal binding occurring at 33 nM, and kinetics for dissociation of bound cAMP were biphasic, indicating the presence of two different binding sites. These observations suggest that residues 1-45 function only in the formation of dimers and that dimerization has little influence on other functional properties of the regulatory subunit. More extensive proteolysis cleaved the monomeric fragment at Lys-311. The fragments resulting from this second cleavage did not dissociate, and the complex inhibited the catalytic subunit in a cAMP-dependent manner.

Rapid identification of NH 2-terminal myristyl peptides by reversed-phase high-performance liquid chromatography

NH 2-terminal myristyl peptides in mixtures of other peptides having the NH 2-terminal blocked with acetyl, formyl or pyroglutamyl groups were selectively determined by high-performance liquid chromatography using a poly(vinyl alcohol) resin column eluted with 50 m M sodium hydrogen carbonate, pH 8.3, and a linear gradient of acetonitrile. This method was applied to the analysis of an NH 2-terminal blocked peptide from a peptic digest of the catalytic subunit of adenosine 3′:5′-phosphate-dependent protein kinase type II from bovine heart, showing that the peptide has a sequence myristyl-Gly-Asn-Ala.

Interaction of guanosine cyclic 3',5'-phosphate dependent protein kinase with lin-benzoadenine nucleotides.

Using the activated cGMP-dependent protein kinase in the presence of the phosphorylatable peptide [[Ala34]histone H2B-(29-35)], we found that lin-benzoadenosine 5'-diphosphate (lin-benzo-ADP) was a competitive inhibitor of the enzyme with respect to ATP with a Ki (22 microM) similar to the Kd (20 microM) determined by fluorescence polarization titrations. The Kd for lin-benzo-ADP determined in the absence of the phosphorylatable peptide, however, was only 12 microM. ADP bound with lower affinity (Ki = 169 microM; Kd = 114 microM). With [Ala34]histone H2B-(29-35) as phosphoryl acceptor, the Km for lin-benzo-ATP was 29 microM, and that for ATP was 32 microM. The Vmax with lin-benzo-ATP, however, was only 0.06% of that with ATP as substrate [0.00623 +/- 0.00035 vs. 11.1 +/- 0.17 mumol (min.mg)-1]. Binding of lin-benzo-ADP to the kinase was dependent upon a divalent cation. Fluorescence polarization revealed that Mg2+, Mn2+, Co2+, Ni2+, Ca2+, Sr2+, and Ba2+ supported nucleotide binding to the enzyme; Ca2+, Sr2+, and Ba2+, however, did not support any measurable phosphotransferase activity. The rank order of metal ion effectiveness in mediating phosphotransferase activity was Mg2+ greater than Ni2+ greater than Co2+ greater than Mn2+. Although these results were similar to those observed with the cAMP-dependent protein kinase [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], major differences in the Vmax with lin-benzo-ATP as substrate and the effect of peptide substrates on nucleotide (both lin-benzo-ADP and ADP) binding were observed.

Simple and rapid identification of phosphorylated peptides from bovine brain myelin basic protein by reversed-phase high-performance liquid chromatography

The phosphorylation sites of the myelin basic protein from bovine brain were determined after phosphorylation with a cyclic 3′:5′-phosphate-dependent protein kinase from the same source. Three phosphorylated peptides were selectively rapidly separated, before and after dephosphorylation, by reversed-phase high-performance liquid chromatography on a styrene 250 column under alkaline conditions. Partial sequencing of the peptides by automated Edman degradation revealed that the serine-115 residue located in the main encephalitogenic determinant of the protein was a phosphorylation site, in addition to the two phosphorylation sites established (threonine-34 and serine-55).

Asynchrony in the expression of guanosine 3′:5′-phosphate-dependent protein kinase by clusters of purkinje cells during the perinatal development of rat cerebellum

The early maturation of Purkinje cells was studied by immunocytochemistry in the rat cerebellum. The antiserum against guanosine 3′:5′-phosphate-dependent protein kinase used in this study has been shown previously to label specifically all Purkinje cells in the adult rat. Immunoreactive Purkinje cells are first observed at embryonic day 17, 2 days after the end of proliferation of this neuronal population. At this time, most of the labeled cells are situated in the subventricular zone, although some immunoreactive Purkinje cells have already reached the cortex. Between embryonic day 17 and birth, four clusters of immunoreactive Purkinje cells appear in each hemicerebellum. Their time course and their pathways of migration to the cortex were followed. The immunoreactive clusters are tailed by a fibre-like immunostained material. The pattern of the migrating clusters at embryonic day 19 is very similar to the pattern of the corticonuclear projection observed at birth. From comparison between sections of embryos processed either for immunocytochemistry or Cresyl Violet staining, it appears that all the Purkinje cells are not immunoreactive. Positive and negative clusters of Purkinje cells are sharply delineated, their cells never mix. Immunopositive and negative clusters of Purkinje cells coexist until postnatal day 3. However, from birth onwards, negative clusters begin progressively in a caudorostral sequence to express guanosine 3′:5′-phosphate-dependent protein kinase and rapidly attain the same level of immunoreactivity as previously labeled clusters. From postnatal day 5 all the Purkinje cells are immunoreactive. It is concluded that a compartmentalization of the cerebellar cortex is present very early and is evidenced by differences in the biochemical maturation of Purkinje cells. The axons of Purkinje cells reach the deep nuclei, following the same pathways as the clusters of Purkinje cells migrating to the cortex. Therefore, the mechanisms regulating the selection of the migratory routes followed by each Purkinje cell cluster are essential for the achievement of the topography of the corticonuclear projection. The level of protein kinase immunoreactivity cannot be taken as an index of the overall maturation of Purkinje cells, because it does not always coincide with the expression of other makers of biochemical and morphological differentiation of these neurons. During the early establishment of the cerebellar maps, an asynchrony in the expression of parts of the same genotype in the Purkinje cells may help in the establishment of ordered connections.

Anatomy of cerebellar Purkinje cells in the rat determined by a specific immunohistochemical marker

In the present study we have used guanosine 3′: 5′-phosphate-dependent protein kinase antiserum, a specific immunohistochemical marker for cerebellar Purkinje cells, [ Lohmann, Walter, Miller, Greengard and De Camilli (1981) Proc. natn. Acad. Sci. U.S.A. 78, 653–657], to carry out a detailed analysis of the architecture and projections of Purkinje cells in the adult rat. We have obtained a novel view of aspects of Purkinje cell morphology that were already known and, in addition, we have provided some new information, in particular on the targets of Purkinje cell axons and their pattern of innervation, and on the morphology and course of Purkinje cell axons. Furthermore, we have found a few cells positive for guanosine 3′: 5′ phosphate-dependent protein kinase which are very similar morphologically to Purkinje cells but are located outside of the cerebellar cortex. A unique feature of Purkinje cells is their peculiar monoplanar shape. Not only do their dendritic arbors lie in planes perpendicular to the major axis of the folia, but their axons, including the collaterals, also travel roughly in the same planes. Thus, Purkinje cells can be imagined as lying in longitudinal sheets radiating from the deep cerebellar nuclei. In these sheets, Purkinje cell axons originating from cells located at different rostrocaudal levels of the cortex converge towards the deep cerebellar nuclei without intersecting each other. It is as a result of this precise organization that Purkinje cell axons reach the deep cerebellar nuclei with a mediolateral and rostrocaudal topology that closely reflects the position of their parent cells in the cerebellar cortex. In the subcortical rays of white matter, Purkinje cell axons are interspersed with other axons, being excluded only from longitudinal strips which correspond to the cerebellar raphes. Upon converging towards the deep cerebellar nuclei they segregate into tracts of white matter that alternate with tracts of white matter from which they are excluded. The great majority of Purkinje cell axons terminate in the deep cerebellar nuclei. Recurrent collaterals terminate in close proximity to the Purkinje cell layer. Dense innervation by these axons is found around large interneurons (Lugaro and Golgi cells) and around the Purkinje cell pinceaux. No direct input of recurrent collaterals to Purkinje cell somata is evident in immunostained material. A substantial number of Purkinje cell axons continue beyond the cerebellar nuclei to innervate nearby regions in the brain stem. The most prominently innervated extracerebellar target region is the dorsal part of the lateral vestibular nucleus, which is as heavily innervated by Purkinje axons as the deep cerebellar nuclei are. All the other major parts of the vestibular formation and some adjacent nuclei (including the parabrachial nuclei, the prepositus hypoglossal nucleus and the nucleus of the solitary tract) are innervated to various degrees by Purkinje cells. In these regions heavily innervated cells are interspersed with cells which receive only a moderate degree of innervation and with cells which apparently lack Purkinje cell inputs. Upon reaching the deep cerebellar nuclei, axons destined to extracerebellar targets deviate from the planes of dendritic arborization of their parent cells. They converge into tight bundles which follow an irregular course and intersect each other to reach their targets. Axons travelling in the same bundle often appear to terminate on the same cell. At all target sites Purkinje axons end as varicose terminals which synapse primarily with the perikarya and proximal dendrites of target cells. On the surface of these cells Purkinje cell terminals are often tightly apposed to form a compact mosaic. Both the course of Purkinje cell axons and their pattern of innervation of target cells are consistent with the possibility that contacts between Purkinje cells and their target neurons in the deep cerebellar nuclei and the brain stem are established early in ontogenesis. Purkinje cell-like cells positive for guanosine 3′:5′-phosphate-dependent protein kinase not located in the cerebellar cortex were found predominantly at the dorsal surface of the brain stem and, in particular, in the cortex of the dorsal cochlear nucleus. Only on exception were they found in the cerebellar medulla or in nearby noncortical regions of the brain stem. While some of these cells might be ectopies, the significance of Purkinje cell-like cells in the dorsal cochlear nucleus, a region strikingly similar in architecture to the cerebellar cortex, remains to be established.

Amino acid sequence of the catalytic subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase.

The 350-residue amino acid sequence of the catalytic subunit of bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase is described. The protein has a molecular weight of 40 862, which includes an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in the S-carboxymethylated protein were cleaved with cyanogen bromide to yield eight primary peptides. These fragments, and subpeptides generated by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and Myxobacter AL-1 protease II, were purified and analyzed to yield the majority of the sequence. The primary peptides were aligned by analyses of overlapping peptides, particularly of methione-containing tryptic peptides generated after in vitro [14C]methyl exchange labeling of methionyl residues in the intact protein.