Proc. Natl. Acad. Sci. USAVol. 81, pp. 4311-4315, July 1984Biochemistry

Ca2+/calmodulin-dependent protein kinases from the neuronalnuclear matrix and post-synaptic density are structurally related

(neuronal plasticity/transcription/chromatin/protein phosphorylation/fodrin)

NAJi SAHYOUN, HARRY LEVINE III, AND PEDRO CUATRECASASDepartment of Molecular Biology, The Wellcome Research Laboratories, 3030 Cornwallis Road, Research Triangle Park, NC 27709

Contributed by Pedro Cuatrecasas, March 29, 1984

ABSTRACT A major Ca2+/calmodulin-dependent pro-tein kinase has been isolated in association with the neuronalnuclear matrix. Nuclear matrix preparations contain highlyphosphorylated polypeptides with Mr values of 50,000 and60,000. These polypeptides were further characterized by pep-tide and phospho peptide mapping, two-dimensional isoelec-trofocusing/NaDodSO4/PAGE, and '25I-labeled calmodulinbinding. The results indicate that the Mr 50,000 and 60,000polypeptides of the nuclear matrix closely resemble the a and.8 subunits, respectively, of the Ca2+/calmodulin-dependentprotein kinase of the post-synaptic density. These findings in-dicate that similar protein kinases mediate the neuronal effectsof Ca2+ at the cytosolic, synaptosomal, and nuclear levels.

We have recently described the occurrence of Ca2+/calmod-ulin-dependent protein phosphorylation in neuronal nuclei(1). The Ca2+/calmodulin-dependent enzyme that mediatesthe phosphorylation appears to be the most abundant nucle-ar protein kinase (1). Nuclear substrates include chromatinproteins which have been implicated in the regulation oftranscriptional events (1). The nuclear enzyme was partiallyextractable with 0.4 M NaCl but largely remained associatedwith the nuclear matrix (1). Molecular characterization ofthis enzyme has been facilitated by the recent purification ofsoluble Ca2+/calmodulin-dependent protein kinases from ratbrain (2-5) that display different substrate specificities butexhibit a similar subunit structure. Two types of polypep-tides are involved: a major a subunit (Mr, 50,000) and a mi-nor ( subunit (Mr 58,000, 60,000); both subunits bind cal-modulin, are autophosphorylated, and yield overlapping pep-tide patterns following protease digestion (2-5). Moreover, asimilar enzyme complex seems to be associated with post-synaptic densities where the major Mr 50,000 polypeptidewas identified as the a subunit of the protein kinase (6, 7).Two polypeptides (Mr, 50,000 and 60,000) in the neuronal

nuclear matrix are heavily phosphorylated in the presence ofCa2+ and calmodulin (1). These polypeptides were comparedwith their presumed counterparts in post-synaptic densitypreparations from rat brain cortices. The corresponding Mr50,000 and 60,000 proteins from the two sources proved tobe similar in several respects. They were both heavily phos-phorylated, bound calmodulin, and yielded comparable tryp-tic peptide maps, as well as phospho peptide fragments fol-lowing digestion with staphylococcal V8 protease. Further-more, two-dimensional isoelectrofocusing/NaDodSO4/PAGE polypeptide analysis revealed that both Ca2+/calmo-dulin-dependent protein kinases migrated largely as a dis-tinct molecular complex with a pI of about 6.7. These resultsdesignate the Ca2+/calmodulin-dependent protein kinase ofneuronal nuclei as a member of a class of enzymes with dif-ferent subcellular localization.

MATERIALS AND METHODSStaphylococcal protease (V8) was purchased from Sigma;calmodulin was also obtained from Sigma and was routinelychecked for purity and biological activity. [y-32PJATP (30-50Ci/mmol; 1 Ci = 37 GBq) was supplied by New EnglandNuclear and 125I-labeled Bolton-Hunter reagent was pro-cured from ICN; Na1251 was provided by Atomic Energy ofCanada (Ottawa, ON). Calmodulin was iodinated with 251-labeled Bolton-Hunter reagent (2000 Ci/mmol) as described(8). Binding of 125I-labeled calmodulin to specific polypep-tides was examined by the gel-overlay method (9) followingprotein resolution by NaDodSO4/PAGE. Phospho peptidemapping using protein digestion with staphylococcal V8 pro-tease was carried out on 13.5% acrylamide gels according tothe method of Cleveland et al. (10). Single proteins were ra-dioiodinated in gel slices using chloramine-T (11), and trypticpeptides thereof were resolved by two-dimensional chroma-tography on silica thin-layer plates (11, 12). NaDodSO4/PAGE was carried out on 7.5% acrylamide gels as describedby Laemmli (13); separation of proteins by two-dimensionalisoelectrofocusing/NaDodSO4/PAGE was carried out ac-cording to the procedure of O'Farrell (14). For autoradiogra-phy, gels were dried and directly exposed to Kodak DEF-5film or to Kodak Royal X-Omat film in the presence of afluorescent screen enhancer. The autoradiograms were ana-lyzed by standardized quantitative scanning densitometry(1); Coomassie blue-stained gels were examined similarly.Protein concentration was determined using Coomassie blueR-250 (15).

Tissues from Sprague-Dawley male rats (100-150 g) wereused throughout. Neuronal nuclei were purified and separat-ed from glial nuclei as detailed (1, 16); liver nuclei were ob-tained as described (17). Nuclear matrix preparations werederived from neuronal nuclei by hypotonic swelling, extrac-tion with 2 M NaCl, and solubilization in Triton X-100 (1,18). Post-synaptic densities were prepared from cortical syn-aptosomes by extraction with Triton X-100 and fractionationon a sucrose-density step gradient (19).

RESULTSThe protein composition of the neuronal nuclear matrix (Fig.1, lane 2) exhibits specific enrichment in several polypep-tides. Notably, three components (Mr 62,000, 65,000,67,000; Fig. 1, lane 2, asterisk) migrate in the region of thethree major polypeptides that have been characterized asmajor constituents of nuclear matrix preparations from liver(18, 20). Other constituents of the neuronal nuclear matrixhave molecular weight values similar to those of severalknown polypeptides (19) from the post-synaptic density (Fig.1, lane 3) such as actin (43,000), fodrin (235,000), and theneurofilament proteins (Mr, 150,000, 68,000). Fig. 2 (lanes 4-6) shows that the neuronal nuclear matrix undergoes markedphosphorylation in the combined presence of Ca2+ and cal-modulin. Major phosphorylated components are evidentwith Mr values of 50,000 and 60,000; the incorporation of 32p

4311

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4312 Biochemistry: Sahyoun et al.

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FIG. 1. Polypeptide composition of the neuronal nuclear matrix,the post-synaptic density, and other subcellular fractions. Lane 1,non-nuclear brain particulate fraction; lane 2, neuronal nuclear ma-trix; lane 3, post-synaptic density; lane 4, liver nuclei; lane 5: neuro-nal nuclei. Numbers on the left represent M, (x 10-3) values of stan-dard human erythrocyte ghost polypeptides; those between lanes 2and 3 correspond to fodrin (235,000), neurofilament proteins(150,000, 68,000), the major Mr 50,000 post-synaptic density protein(50,000), and actin (43,000). The asterisk highlights three polypep-tides whose mobilities are similar to those of the major nuclear ma-trix proteins from liver.

into the Mr 50,000 moiety exceeds that in the Mr 60,000 moi-ety by about 2-fold. The phosphorylation of both compo-nents depends on the concentration of calmodulin with anEC50 of 40 nM at 30 1kM CaC12. Interestingly, cortical post-synaptic density preparations contain two polypeptides withidentical molecular weight values and a 3 P-labeling profilesimilar to that observed in the nuclear matrix (Fig. 2, lanes1-3). Recently, the 50,000 and 60,000 polypeptides frompost-synaptic density preparations were identified as the a

and 8 subunits, respectively, of a prominent brain Ca2+/cal-modulin-dependent protein kinase (6, 7) that had been puri-fied earlier in the soluble state from brain cytosol (2-4). Con-sequently, we investigated the possibility that the Mr 50,000and 60,000 phosphorylated components from the neuronalnuclear matrix represent the a and 18 subunits, respectively,of a nuclear form of the Ca2+/calmodulin-dependent proteinkinase.Employing the gel-overlay method, it was shown (9) that

I251-labeled calmodulin bound to the Mr 50,000 major post-synaptic density protein. Likewise, both the a and 1 sub-units of the soluble brain Ca2+/calmodulin-dependent pro-tein kinase manifested 125I-labeled calmodulin binding (3).Fig. 3 (lane 2) shows that the Mr 50,000 and 60,000 polypep-tides are major constituents of the neuronal nuclear matrixthat bind 125I-labeled calmodulin in a reaction that is inhibit-ed by 100 ,uM trifluoperazine. The Mr 50,000 componentbinds about 4 times more radiolabeled calmodulin than theMr 60,000 component. The pattern of 125I-labeled calmodulinbinding to the two nuclear polypeptides closely approxi-mates the binding pattern displayed by post-synaptic density

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FIG. 2. Ca2" and calmodulin requirements of endogenous pro-tein phosphorylation in the neuronal nuclear matrix and the post-synaptic density. The phosphorylating reaction mixture (100 /d)contained 50 AtM ATP, 1 tLCi of [y-32P]ATP, 7 mM MgCl2, 50 mMTris HCl (AH 7.7), and 20 Fig of the enzyme preparation. EG, 1 mMEGTA; Ca +, 100 AM CaC12; CM, calmodulin at 1 pg/ml; NM, nu-clear matrix; PSD, post-synaptic density. Reactions were carriedout at 30'C for 5 min and terminated by addition of NaDodSO4-con-taining buffer. Protein phosphorylation was assessed by gel electro-phoresis followed by autoradiography. Mr values on the right denotethe major phosphorylated components (50,000, 60,000). 32p incorpo-ration into the nuclear matrix components represents about 10%o ofthe corresponding incorporation in the post-synaptic density. Ac-cordingly, the autoradiograms for the nuclear matrix were exposedfor a longer period of time.

preparations (lane 3). Furthermore, both intact neuronal nu-clei (lane 5) and matrix preparations therefrom result in simi-lar patterns of 1251-labeled calmodulin binding, indicatingthat the matrix calmodulin-binding sites also comprise majorcalmodulin-binding polypeptides in whole nuclei. In con-trast, liver nuclei (lane 4) reveal minimal calmodulin bindingin the Mr 50,000 or 60,000 region; this observation agreeswith our previous report that protein phosphorylation in liv-er nuclei lacks Ca2+/calmodulin sensitivity (1). Fig. 3 re-veals that other components of potential interest bind calmo-dulin. Thus, a prominent Coomassie blue-staining Mr235,000 polypeptide in the neuronal nuclear matrix (Fig. 1,lane 2) exhibits marked association with calmodulin (lane 2).Calmodulin binding in the same region is also present inwhole nuclei (lane 5) as well as in non-nuclear neuronal par-ticulate material (lane 1) and in post-synaptic densities (lane3). Recently, a Mr 235,000 polypeptide from post-synapticdensities was identified as fodrin (21) or brain spectrin. This

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FIG. 3. Binding of ".'I-labeled calmodulin to resolved polypeptides of the neuronal nuclear matrix and other relevant subcellular structures."5I-labeled calmodulin (0.1 ,uCi/ml) binding was assessed employing the gel-overlay method (9) with 100 LM CaCl2 in the absence (left) orpresence (right) of 100 ,uM trifluoperazine. Lane 1, non-nuclear brain particulate fraction; lane 2, neuronal nuclear matrix; lane 3, post-synapticdensity; lane 4, liver nuclei; lane 5, neuronal nuclei. Selected Mr (x 10-) values are indicated between lanes 2 and 3 (Left).

suggests that fodrin or a similar protein also may be a majorcalmodulin-binding component in the neuronal nuclear ma-trix, whole neuronal nuclei, and whole liver nuclei. In addi-tion, three low Mr polypeptides (Mr, 25,000-30,000) in neu-ronal and liver nuclei display remarkable calmodulin bindingwhose functional significance has not been ascertained.The Mr 50,000 and 60,000 components of the neuronal nu-

clear matrix and of the post-synaptic density were maximallyphosphorylated and then subjected to phospho peptide map-ping (Fig. 4). Digestion with staphylococcal V8 protease pro-duces closely matching phospho peptide fragments for theMr 60,000 component from the two sources (lanes 1 and 5 vs.3 and 7). The phospho peptide maps from the Mr 50,000 moi-eties (lanes 2 and 6 vs. 4 and 8) show major overlapping frag-ments but some significant differences as well. These differ-ences may result from a real variation in the primary proteinstructure or might reflect altered sites of phosphorylation.To examine these alternatives, tryptic maps of the radioiodi-nated Mr 50,000 polypeptides were generated. The selectionof the nuclear Mr 50,000 polypeptide was based on severalcriteria. Neuronal, but not liver, nuclear matrix preparationsconsistently displayed the presence of a Mr 50,000 polypep-tide that stained clearly with Coomassie blue at 10o of therelative staining intensity of the post-synaptic density coun-terpart (Fig. 1, lane 2, arrow) and comigrated with the corre-sponding phosphorylated polypeptide (Fig. 2). Furthermore,hyperphosphorylation of nuclear matrix preparations led toan apparent increase in the Mr value of about 3,000 (data notshown); the ot subunit of the soluble Ca2+/calmodulin-de-pendent protein kinase undergoes a corresponding decreasein mobility (3). These observations suggested that the Mr50,000 Coomassie blue-staining band might represent the asubunit of the nuclear matrix-bound protein kinase. Fig. 5illustrates the considerable similarity between the two-di-mensional tryptic maps of the radioiodinated major Mr50,000 component of the post-synatic density and of its puta-tive counterpart in the nuclear matrix. Additional evidencefor the structural relationship between the nuclear and thepost-synaptic density enzymes derives from isoelectrofo-

cusing/NaDodSO4/PAGE analysis (Fig. 6). The Mr 50,000and 60,000 phospho proteins from both sources apparentlymigrate as a single molecular complex with an approximate

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FIG. 4. Phospho peptide mapping of the Mr 50,000 and 60,000polypeptides. Neuronal nuclear matrix and post-synaptic densitypreparations were maximally phosphorylated using 50 mM Tris * HC(pH 7.7) in the presence of 0.5 mM ATP, 5 uCi of [y-32P]ATP, 10mM MgCl2, 100 ,uM CaCl2, and calmodulin at 1 ,ug/ml. Protein sam-ples (10 Ag) were resolved by NaDodSO4/PAGE, and the regions ofthe Mr 50,000 and 60,000 phosphorylated polypeptides were excisedand subjected to peptide mapping according to the method of Cleve-land et al. (10) in the presence of 25 ng (lanes 1-4) or 100 ng (lanes 5-8) of staphylococcal V8 protease. Lanes 2 and 6, Mr 50,000 polypep-tide from the post-synaptic density; lanes 1 and 5, Mr 60,000 poly-peptide from the post-synaptic density; lanes 4 and 8, Mr 50,000polypeptide from the nuclear matrix; lanes 3 and 7, Mr 60,000 poly-peptide from the nuclear matrix.

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FIG. 5. Tryptic map of radiolabeled Mr 50,000 polypeptides from the nuclear matrix and post-synaptic density. (A and B) Autoradiograms ofpost-synaptic density and nuclear matrix, respectively. (C) Diagrammatic representation of the position of the autoradiographic spots regardlessof their intensity. Open spots are common to both preparations, hatched spots are unique to the post-synaptic density subunit, and closed spotsare present exclusively in the nuclear matrix subunit. The silica plates were developed with an organic phase of n-butanol/acetic acid/H20,4:1:5 (vol/vol) in the first dimension followed by n-butanol/pyridine/acetic acid/H20, 32.5:25:5:20 (vol/vol) in the second dimension.

pl of 6.7. The partial smearing of both proteins agrees withprevious analysis of the major Mr 50,000 protein of the post-synaptic density (12). Other phosphorylated components,especially in the high molecular weight region, appear to co-align with the Mr 50,000-60,000 complex (Fig. 6, asterisk)implicating other polypeptides in the formation of the appar-ent molecular complex. However, an additional phosphoryl-ated Mr 50,000 polypeptide appears at about pl 4.7 (Fig. 6,arrow), joined by a faint autoradiographic smear to the Mr50,000 moiety at pl 6.7. The pl 4.7 material may, therefore,represent a dissociated form of the Mr 50,000 subunit.

DISCUSSIONNeuronal nuclei contain a major endogenous Ca2+/calmodu-lin-dependent protein kinase that phosphorylates a specificsubset of nuclear proteins (1). Prominent among the nuclearsubstrates is a component which comigrates with HMG17, achromatin protein that seems to be associated with activegenes (1, 22-26). The association of this enzyme with thenuclear matrix is also consistent with its putative involve-ment in regulating transcriptional processes. This suggestionis based on substantial evidence attributing a role for the nu-clear matrix in transcriptional mechanisms (27, 28). This evi-dence has been further strengthened by recent reports of thepreferential association of active DNA with the nuclear ma-trix (29, 30) and of the inhibition of ribonucleoprotein trans-port by antibodies against lamin B (31), a major constituentof the pore-lamina complex. The age and tissue dependenceof the enzyme levels also suggest its involvement in nuclearevents relevant to tissue differentiation (1).

Extranuclear Ca2+/calmodulin-dependent protein kinaseshave been purified from brain cytosol (2-5) and identifiedwith' the major Mr 50,000 component of the post-synapticdensity (6, 7). Three brain enzymes seem to possess a simi-lar, if not identical, subunit structure. The subunits are orga-

nized in a macromolecular complex containing a (Mr 50,000)and (3 (Mr 58,000, 60,000) components with a preponderancein a ratio of 3:1 or 5:1 (3). The purified soluble enzymes havesimilar calculated Mr values of 600,000-650,000 but differ insubstrate specificity with preference for synapsin 1 (3) or fortubulin (4). The post-synaptic density enzyme appears toshare an a,/3 structure similar to that of the cytosolic synap-sin I kinase (3).

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Proc. Natl. Acad Sci. USA 81 (1984)

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Proc. NatL. Acad. Sci. USA 81 (1984) 4315

Here, we have examined the possibility that the major50,000 and 60,000 phosphorylated polypeptides of the neuro-nal nuclear matrix represent, respectively, the a and /3 sub-units of a Ca2+/calmodulin-dependent protein kinase. Thedata show that the molecular structure of the nuclear en-zyme resembles closely that of the post-synaptic density andtherefore of the soluble synapsin I kinase as well. Supportiveevidence includes Ca2+/calmodulin-stimulated phosphoryl-ation, tryptic maps of the a subunits, phospho peptide mapsof the a and /3 subunits, 125I-labeled calmodulin binding, be-havior on two-dimensional isoelectrofocusing/NaDodSO4/PAGE, and the apparent stoichiometry of the a,/3 compo-nents. The peptide and phospho peptide maps manifestmany similarities between the nuclear and post-synapticdensity enzymes, but a few peptide and phospho peptidefragments appear to be specific to the enzyme source. Thesediscrepancies may result from contaminating polypeptidesbut alternatively, may be related to the subcellular localiza-tion of the enzyme. The designation of the Mr 50,000 and60,000 nuclear polypeptides as major calmodulin-binding en-tities may also account for the recent histochemical evidencethat calmodulin is highly concentrated in the adult rat neuro-nal nucleus (32).The occurrence of homologous Ca2-/calmodulin-depen-

dent protein kinases in different neuronal organelles mayprovide a powerful mechanism for ubiquitous and coordinat-ed regulation of neuronal phosphorylation by altered Ca21levels. The identification of a nuclear subclass of this Ca2 -sensitive protein kinase may permit the inte ration of regula-tory nuclear events with non-nuclear Ca +-sensitive pro-cesses. The mechanism by which neuronal Ca2+/calmodu-lin-dependent protein kinases are restricted to differentsubcellular compartments remains to be elucidated.

We would like to thank Mr. Duane Bronson for his expert assis-tance.

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