Specific binding of a 14-3-3 protein to autophosphorylated WPK4, an SNF1-related wheat protein kinase, and to WPK4-phosphorylated nitrate reductase

WPK4 is a wheat protein kinase related to the yeast protein kinase SNF1, which plays a role in catabolite repression. To identify proteins involved in signal transduction through WPK4, we performed yeast two-hybrid screens and isolated two cDNA clones designated as TaWIN1 and TaWIN2. Both encode 14-3-3 proteins that, upon autophosphorylation, bind the C-terminal regulatory domain of WPK4. Mutational analysis through amino acid substitution revealed that TaWIN1 and TaWIN2 primarily bind WPK4 through phosphoserines at the positions 388 and 418, both located in the C-terminal region. Mutations in the conserved residues of the TaWIN1 amphipathic groove impaired the ability of TaWIN1 to bind to WPK4. A screen for in vitro phosphorylation of proteins involved in nutrient metabolism revealed a putative WPK4 substrate, nitrate reductase; its hinge 1 region was efficiently phosphorylated by WPK4. Subsequent far Western blots showed that it specifically bound TaWIN1. Since nitrate reductase has been shown to be inactivated by phosphorylation upon 14-3-3 binding, the present findings strongly suggest that WPK4 is the protein kinase responsible for controlling the nitrogen metabolic pathway, assembling the nitrate reductase and 14-3-3 complex through its phosphorylation specificity.     

Review: model peptides and the physicochemical approach to beta-amyloids

beta-Amyloid peptides are the main protein components of neuritic plaques and may be important in the pathogenesis of Alzheimer's Disease. The determination of the structure of beta-amyloid fibrils poses a challenge because of the limited solubility of beta-amyloid peptides and the noncrystalline nature of fibrils formed from these peptides. In this paper, we describe several physicochemical approaches which have been used to examine fibrils and the fibrillogenesis of peptide models of beta-amyloid. Recent advances in solid state NMR, such as the DRAWS pulse sequence, have made this approach a particularly attractive one for peptides such as beta-amyloid, which are not yet amenable to high-resolution solution phase NMR and crystallography. The application of solid state NMR techniques has yielded information on a model peptide comprising residues 10-35 of human beta-amyloid and indicates that in fibrils, this peptide assumes a parallel beta-strand conformation, with all residues in exact register. In addition, we discuss the use of block copolymers of Abeta peptides and polyethylene glycol as probes for the pathways of fibrillogenesis. These methods can be combined with other new methods, such as high-resolution synchrotron X-ray diffraction and small angle neutron and X-ray scattering, to yield structural data of relevance not only to disease, but to the broader question of protein folding and self-assembly.     

Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions

A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP(2)-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP(2). Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP(2) laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP(2) in the plasma membrane.     

Targeted control of kinetics of beta-amyloid self-association by surface tension-modifying peptides

Brain tissue from Alzheimer's patients contains extracellular senile plaques composed primarily of deposits of fibrillar aggregates of beta-amyloid peptide. beta-Amyloid aggregation is postulated to be a major factor in the onset of this neurodegenerative disease. Recently proposed is the hypothesis that oligomeric intermediates, rather than fully formed insoluble fibrils, are cytotoxic. Previously, we reported the discovery of peptides that accelerate beta-amyloid aggregation yet inhibit toxicity in vitro, in support of this hypothesis. These peptides contain two domains: a recognition element designed to bind to beta-amyloid and a disrupting element that alters beta-amyloid aggregation kinetics. Here we show that the aggregation rate-enhancing activity of the disrupting element correlates strongly with its ability to increase surface tension of aqueous solutions. Using the Hofmeister series as a guide, we designed a novel peptide with terminal side-chain trimethylammonium groups in the disrupting domain. The derivatized peptide greatly increased solvent surface tension and accelerated beta-amyloid aggregation kinetics by severalfold. Equivalent increases in surface tension in the absence of a recognition domain had no effect on beta-amyloid aggregation. These results suggest a novel strategy for targeting localized changes in interfacial energy to specific proteins, as a way to selectively alter protein folding, stability, and aggregation.     

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

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

Rim,a component of the presynaptic active zone and modulator of exocytosis,binds 14-3-3 through its N terminus

Rim1, a brain-specific Rab3a-binding protein, localizes to the presynaptic cytomatrix and plays an important role in synaptic transmission and synaptic plasticity. Rim2, a homologous protein, is more ubiquitously expressed and is found in neuroendocrine cells as well as in brain. Both Rim1 and Rim2 contain multiple domains, including an N-terminal zinc finger, which in Rim1 strongly enhances secretion in chromaffin and PC12 cells. The yeast two-hybrid technique identified 14-3-3 proteins as ligands of the N-terminal domain. In vitro protein binding experiments confirmed a high-affinity interaction between the N terminus of Rim1 and 14-3-3. The N-terminal domain of Rim2 also bound 14-3-3. The binding domains were localized to a short segment just C-terminal to the zinc finger. 14-3-3 proteins bind to specific phosphoserine residues. Alkaline phosphatase treatment of N-terminal domains of Rim1 and Rim2 almost completely inhibited the binding of 14-3-3. Two serine residues in Rim1 (Ser-241 and Ser-287) and one serine residue in Rim2 (Ser-335) were required for 14-3-3 binding. Incubation with Ca2+/calmodulin-dependent protein kinase II greatly stimulated the interaction of recombinant N-terminal Rim but not the S241/287A mutant with 14-3-3, again indicating the importance of the phosphorylation of these residues for the binding. Rabphilin3, another Rab3a effector, also bound 14-3-3. Serine-to-alanine mutations identified Ser-274 as the likely phosphorylated residue to which 14-3-3 binds. Because the phosphorylation of this residue had been shown to be stimulated upon depolarization in brain slices, the interaction of 14-3-3 with Rabphilin3 may be important in the dynamic function of central nervous system neurons.     

Peptides that mimic the pseudosubstrate region of protein kinase C bind to acidic lipids in membranes.

The cytoplasmic form of protein kinase C (PKC) is inactive, probably because the pseudosubstrate region in its regulatory domain blocks the substrate-binding site in its kinase domain. Calcium ions cause a translocation to the membrane: maximum activation requires a negative lipid such as phosphatidylserine (PS) and the neutral lipid diacylglycerol (DAG) but the mechanism by which PS and DAG activate PKC is unknown. Pseudosubstrate region 19-36 of PKC-beta has six basic and one acidic amino acids and region 19-29 has five basic and no acidic amino acids. Since any binding of basic residues in the pseudosubstrate region to acidic lipids in the membrane should stabilize the active form of PKC, we studied how peptides with amino acids equivalent to residues 19-36 and 19-29 of PKC-beta bound to phospholipid vesicles. We made equilibrium dialysis, filtration, and electrophoretic mobility measurements. The fraction of bound peptide is a steep sigmoidal function of the mol fraction of negative lipid in the membrane, as predicted from a simple theoretical model that assumes the basic residues provide identical independent binding sites. The proportionality constant between the number of bound peptides/area and the concentration of peptide in the bulk aqueous phase is 1 micron for a membrane with 25% negative lipid formed in 0.1 M KCl. Equivalently, the association constant of the peptide with the membrane is 10(4) M-1, or the net binding energy is 6 kcal/mol. Thus the interaction of basic residues in the pseudosubstrate region with acidic lipids in the membrane could provide 6 kcal/mol free energy towards stabilizing the active form of PKC.     

Apolipoprotein E and peptide mimetics modulate inflammation by binding the SET protein and activating protein phosphatase 2A

The molecular mechanism by which apolipoprotein E (apoE) suppresses inflammatory cytokine and NO production is unknown. Using an affinity purification approach, we found that peptide mimetics of apoE, derived from its receptor binding domain residues 130-150, bound to the SET protein, which is a potent physiological inhibitor of protein phosphatase 2A (PP2A). Both holo-apoE protein and apoE-mimetic peptides bound to the C-terminal region of SET, which is then associated with an increase in PP2A-mediated phosphatase activity. As physiological substrates for PP2A, the LPS-induced phosphorylation status of signaling MAPK and Akt kinase is reduced following treatment with apoE-mimetic peptides. On the basis of our previous report, in which apoE-mimetic peptides reduced I-κB kinase and NF-κB activation, we also demonstrate a mechanism for reduced production of inducible NO synthase protein and its NO product. These data provide evidence for a novel molecular mechanism by which apoE and apoE-mimetic peptides antagonize SET, thereby enhancing endogenous PP2A phosphatase activity, which reduces levels of phosphorylated kinases, signaling, and inflammatory response.     

Convergent actions of I kappa B kinase beta and protein kinase C delta modulate mRNA stability through phosphorylation of 14-3-3 beta complexed with tristetraprolin

Regulation of gene expression at the level of mRNA stability is a major topic of research; however, knowledge about the regulatory mechanisms affecting the binding and function of AU-rich element (ARE)-binding proteins (AUBPs) in response to extracellular signals is minimal. The beta1,4-galactosyltransferase 1 (beta4GalT1) gene enabled us to study the mechanisms involved in binding of tristetraprolin (TTP) as the stability of its mRNA is regulated solely through one ARE bound by TTP in resting human umbilical vein endothelial cells. Here, we provide evidence that the complex formation of TTP with 14-3-3beta is required to bind beta4GalT1 mRNA and promote its decay. Furthermore, upon tumor necrosis factor alpha stimulation, the activation of both Ikappabeta kinase and protein kinase Cdelta is involved in the phosphorylation of 14-3-3beta on two serine residues, paralleled by release of binding of TTP and 14-3-3beta from beta4GalT1 mRNA, nuclear sequestration of TTP, and beta4GalT1 mRNA stabilization. Thus, a key mechanism regulating mRNA binding and function of the destabilizing AUBP TTP involves the phosphorylation status of 14-3-3beta.     

Poly-L-proline type II peptide mimics as probes of the active site occupancy requirements of cGMP-dependent protein kinase.

Based on the X-ray crystal structure of cAMP-dependent protein kinase (PKA) with the endogenous inhibitor PKI and the X-ray crystal structure of cyclin-dependent kinase 2 (CDK2) with a substrate peptide, a proposal is put forth that some protein kinases bind peptide substrates in their active sites in the poly-L-proline type II (PPII) conformation. In this work, PPII peptide mimics are evaluated as pseudosubstrate inhibitors of cGMP-dependent protein kinase (PKG) to explore if PKG also binds peptide substrates in the PPII conformation. Inhibition data of our PPII mimetics provide evidence that the P-1, P-2, and P-3 residues of substrate peptides bind in the PPII conformation (phi approximately -75 degrees, psi approximately 145 degrees). In addition, the inhibition data also suggest that the P-1, P-2, and P-3 residues in substrate peptides bind with a gauche(-) chi1 angle.     

Multiple forms of copper (II) co-ordination occur throughout the disordered N-terminal region of the prion protein at pH 7.4

Although the physiological function of the prion protein remains unknown, in vitro experiments suggest that the protein may bind copper (II) ions and play a role in copper transport or homoeostasis in vivo. The unstructured N-terminal region of the prion protein has been shown to bind up to six copper (II) ions, with each of these ions co-ordinated by a single histidine imidazole and nearby backbone amide nitrogen atoms. Individually, these sites have micromolar affinities, which is weaker than would be expected of a true cuproprotein. In the present study, we show that with subsaturating levels of copper, different forms of co-ordination will occur, which have higher affinity. We have investigated the copper-binding properties of two peptides representing the known copper-binding regions of the prion protein: residues 57-91, which contains four tandem repeats of the octapeptide GGGWGQPH, and residues 91-115. Using equilibrium dialysis and spectroscopic methods, we unambiguously demonstrate that the mode of copper co-ordination in both of these peptides depends on the number of copper ions bound and that, at low copper occupancy, copper ions are co-ordinated with sub-micromolar affinity by multiple histidine imidazole groups. At pH 7.4, three different modes of copper co-ordination are accessible within the octapeptide repeats and two within the peptide comprising residues 91-115. The highest affinity copper (II)-binding modes cause self-association of both peptides, suggesting a role for copper (II) in controlling prion protein self-association in vivo.     

Memapsin 2 (beta-secretase) cytosolic domain binds to the VHS domains of GGA1 and GGA2: implications on the endocytosis mechanism of memapsin 2

Memapsin 2, or beta-secretase, is a membrane-anchored aspartic protease that initiates the cleavage of beta-amyloid precursor protein (APP) leading to the production of beta-amyloid peptide in the brain and the onset of Alzheimer's disease. Memapsin 2 and APP are both endocytosed into endosomes for cleavage. Here we show that the cytosolic domain of memapsin 2, but not that of memapsin 1, binds the VHS domains of GGA1 and GGA2. Gel-immobilized VHS domains of GGA1 and GGA2 also bound to full-length memapsin 2 from cell mammalian lysates. Mutagenesis studies established that Asp(496), Leu(499) and Leu(500) were essential for the binding. The spacing of these three residues in memapsin 2 is identical to those in the cytosolic domains of mannose-6-phosphate receptors, sortilin and low density lipoprotein receptor-related protein 3. These observations suggest that the endocytosis and intracellular transport of memapsin 2, mediated by its cytosolic domain, may involve the binding of GGA1 and GGA2.     

Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-ζ chain

A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated ζ-chain (TCR-ζ) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (P13K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-ζ may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering. © 1996 Wiley-Liss, Inc.     

Heparin-binding histidine and lysine residues of rat selenoprotein P

Selenoprotein P is a plasma protein that has oxidant defense properties. It binds to heparin at pH 7.0, but most of it becomes unbound as the pH is raised to 8.5. This unusual heparin binding behavior was investigated by chemical modification of the basic amino acids of the protein. Diethylpyrocarbonate (DEPC) treatment of the protein abolished its binding to heparin. DEPC and [(14)C]DEPC modification, coupled with amino acid sequencing and matrix-assisted laser desorption ionization-time of flight mass spectrometry of peptides, identified several peptides in which histidine and lysine residues had been modified by DEPC. Two peptides from one region (residues 80-95) were identified by both methods. Moreover, the two peptides that constituted this sequence bound to heparin. Finally, when DEPC modification of the protein was carried out in the presence of heparin, these two peptides did not become modified by DEPC. Based on these results, the heparin-binding region of the protein sequence was identified as KHAHLKKQVSDHIAVY. Two other peptides (residues 178-189 and 194-234) that contain histidine-rich sequences met some but not all of the criteria of heparin-binding sites, and it is possible that they and the histidine-rich sequence between them bind to heparin under some conditions. The present results indicate that histidine is a constituent of the heparin-binding site of selenoprotein P. The presence of histidine, the pK(a) of which is 7.0, explains the release of selenoprotein P from heparin binding as pH rises above 7.0. It can be speculated that this property would lead to increased binding of selenoprotein P in tissue regions that have low pH.     

The Cdc42 binding and scaffolding activities of the fission yeast adaptor protein Scd2

The small GTP-binding protein Cdc42, the guanine nucleotide exchange factor Scd1, the p21-activated kinase Shk1, and the adaptor protein Scd2 are involved in the Cdc42-dependent signaling cascade in fission yeast. In the present study, we analyzed the Cdc42 binding and scaffolding activities of Scd2 by co-precipitation assays. We found that two SH3-containing regions, amino acid residues 1-87 (CB1 (Cdc42-binding region 1)) and 110-266 (CB2), of Scd2 can bind to the GTP-bound form of Cdc42. CB2 is cryptic because of the intramolecular binding between the SH3 domain in CB2 (SH3(C)) and the PX domain and binds to Cdc42 only when the Scd2 PB1 domain binds to the PC motif-containing region (residues 760-872) of Scd1. This CB2.Cdc42 association, which would stabilize the open configuration of Scd2, enables the SH3(C) domain to bind to the polyproline motif of Shk1. We also found that the GTP-bound form of Cdc42 binds to the CRIB motif of Shk1 more strongly than to Scd2. Thus, Scd2 functions as a scaffold to form a protein complex, and the GTP-bound Cdc42 might be transferred effectively from the upstream activator Scd1 to the downstream effector Shk1 via Scd2.     

An FT-IR study of the beta-amyloid conformation: standardization of aggregation grade

The aggregation of beta-amyloid peptides is very important for their neurotoxic effect; standardization of the aggregation grade is necessary for biological experiments. Measurement of aggregation with physicochemical methods is a difficult task. The present work revealed that FT-IR can be used for studying the aggregation properties of beta-amyloid peptides and the effects of environmental variables (solvent, pH, ions, and temperature) on aggregation. In dimethyl sulfoxide or hexafluoroisopropanol, amyloid peptides are in a monomeric state; on dilution with phosphate buffer just before measurement is made, aggregation begins. A detailed two-dimensional FT-IR correlation spectroscopic study was made of the conformational transitions that occur during the aggregation of beta-amyloid peptides. Two processes (random/helix-to-beta-sheet and aggregation of beta-sheets) and multiple conformational states were observed before the most stable form was attained. beta-Amyloid peptides undergo decomposition in basic buffers containing Ca(2+); this process should be avoided during aging experiments.     

Mechanistic studies of cAMP-dependent protein kinase action

The details of the process by which protein kinase catalyzes phosphoryl group transfers are beginning to be understood. Early work that explored the primary specificity of cAMP-dependent protein kinase action enabled the synthesis of small peptide substrates for the enzyme. Enzyme-peptide interactions seem simpler to understand than protein-protein interactions, so peptide substrates have been used in most protein kinase studies. In most investigations the kinetics for the phosphorylation of small peptides have been interpreted as being consistent with mechanisms which do not invoke phospho-enzyme intermediates (see, for example, Bolen et al.). Protein kinase has been shown to bind two metal ions in the presence of a nucleotide. Using magnetic resonance techniques the binding of these ions has been utilized to elucidate the conformation of nucleotide and peptide substrates or inhibitors when bound in the enzymic active site. Also, two new peptides with the form Leu-Arg-Arg-Ala-Ser-Y-Gly, where Y was either Pro or (N-methyl)Leu, were synthesized and found not to be substrates, within the limits of detection, for protein kinase. The striking lack of affinity that protein kinase has for such peptides which are unlikely to form a beta 3-6 turn has not been reported before. Our results may indicate that this type of turn is a requirement for protein kinase catalyzed phosphorylation or that these peptides lack the ability to form a particular hydrogen bond with the enzyme. Magnetic resonance techniques have indicated that the distance between the phosphorous in the gamma-phosphoryl group of MgATP and the hydroxyl oxygen of serine in the peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly is 5.3 +/- 0.7 A. This, together with certain kinetic evidence, suggests that the mechanism by which protein kinase catalyzes phosphoryl group transfer has considerable dissociative character. Chemical modifications, including one using a peptide-based affinity label, have identified two residues at or near the active site, lysine-72 and cysteine 199. While neither of these groups has been shown to be catalytically essential, similar studies may help to identify groups that are directly involved in the catalytic process. Finally, a spectrophotometric assay for cAMP-dependent protein kinase has been described. Using this assay the preliminary results of an in-depth study of the pH dependence of protein kinase catalyzed phosphoryl group transfer have been obtained. This study shall aid in the identification of active site residues and should contribute to the elucidation of the enzyme's catalytic mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Type II regulatory subunit dimerization determines the subcellular localization of the cAMP-dependent protein kinase

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of dimeric regulatory subunits (RII) to anchoring proteins. Cytoskeletal localization occurs through RII dimer interaction with the PKA substrate molecule microtubule-associated protein 2 (MAP2). RII alpha deletion mutants and RII alpha/endonexin chimeras retained MAP2 binding activity if they contained the first 79 residues of the molecule. Disruption of RII alpha dimerization always prevented MAP2 interaction because 1) RII delta 1-14 (an amino-terminal deletion mutant lacking residues 1-14) was unable to bind MAP2 or form dimers, and 2) a modified RII alpha monomer including residues 1-14 did not bind MAP2. Chimeric proteins containing the first 30 residues of RII alpha fused to endonexin II formed dimers but did not bind MAP2. This suggested other side-chains between residues 30-79 also participate in MAP2 interaction. Peptide studies indicate additional contact with MAP2 may occur through an acidic region (residues 68-82) close to the RII autoinhibitor domain. Therefore, anchored PKA holoenzyme topology may position the catalytic subunit and MAP2 as to allow its preferential phosphorylation upon kinase activation.