©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphorylation and Activation of Ca-Calmodulin-dependent Protein Kinase IV by Ca-Calmodulin-dependent Protein Kinase Ia Kinase
PHOSPHORYLATION OF THREONINE 196 IS ESSENTIAL FOR ACTIVATION (*)

Michele A. Selbert (1), Kristin A. Anderson (2), Qi-Hui Huang (2), Elaine G. Goldstein (1), Anthony R. Means (2), Arthur M. Edelman (1)(§)

From the  (1)Department of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York 14214 and the (2)Department of Pharmacology, Duke University, Durham, North Carolina 27710

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Purified pig brain Ca-calmodulin (CaM)-dependent protein kinase Ia kinase (Lee, J. C., and Edelman, A. M.(1994) J. Biol. Chem. 269, 2158-2164) enhances, by up to 24-fold, the activity of recombinant CaM kinase IV in a reaction also requiring Ca-CaM and MgATP. The addition of brain extract, although capable of activating CaM kinase IV by itself, provides no further activation beyond that induced by purified CaM kinase Ia kinase, consistent with the lack of a requirement of additional components for activation. Activation is accompanied by the development of significant (38%) Ca-CaM-independent CaM kinase IV activity. In parallel fashion to its activation, CaM kinase IV is phosphorylated in a CaM kinase Ia kinase-, Ca-CaM-, and MgATP-dependent manner. Phosphorylation occurs on multiple serine and threonine residues with a Ser-P:Thr-P ratio of 3:1. The identical requirements for phosphorylation and activation and a linear relationship between extent of phosphorylation of CaM kinase IV and its activation state indicate that CaM kinase IV activation is induced by its phosphorylation. Replacement of Thr-196 of CaM kinase IV with a nonphosphorylatable alanine by site-directed mutagenesis abolishes both the phosphorylation and activation of CaM kinase IV, demonstrating that Thr-196 phosphorylation is essential for activation.


INTRODUCTION

Ca-calmodulin (CaM)()-dependent protein kinases (CaM kinases) have long been known to mediate effects of elevated intracellular Ca on cellular functions such as glycogen metabolism, muscle contraction, and neurotransmitter synthesis and release. New observations suggest that, in addition, CaM kinases are involved in Ca-dependent regulation of translational rates, of the transcription of specific genes, and of cell cycle events (for reviews, see (1) ). The CaM kinases constitute a family of related enzymes that includes, among its better-characterized members, phosphorylase kinase, myosin light chain kinase, and CaM kinase II (reviewed in (2) and (3) ). Recently, a significant amount of information about CaM kinase structure and function has been added with the successful purification, characterization, and/or cloning of CaM kinases I(4, 5, 6, 7, 8, 9, 10) , III(11, 12) , and IV(13, 14, 15, 16, 17, 18, 19, 20, 21) .

A critical issue in the study of CaM kinase regulation is the molecular mechanism by which these kinases are activated in response to elevations in Ca concentrations. The currently accepted paradigm is a conformational change induced by the stoichiometric binding of Ca-CaM, which in turn results in decreased interaction of an autoinhibitory domain with the active site, thereby permitting substrate accessibility. This mechanism is ideally suited to the decoding of rapid Ca transients provoked by extracellular signals and is well understood at the molecular level primarily through studies with myosin light chain kinase and CaM kinase II(22, 23, 24, 25, 26, 27, 28) . Evidence has now accumulated, based on studies with CaM kinase I and CaM kinase IV, for an important mechanism of CaM kinase activation in addition to Ca-CaM-induced relief from autoinhibition.

It was reported that Ca-CaM-dependent autophosphorylation of purified rat brain CaM kinase I (a-isoform) is accompanied by a dramatic (>10-fold) potentiation of activity and that an activator exists that is separable from CaM kinase Ia during the final steps of the latter's purification(5, 6) . These observations were confirmed for CaM kinase V(29) , a CaM kinase I isoform(9) . The successful purification of CaM kinase Ia activator from pig brain was recently reported(30) . In the presence of the purified activator, CaM kinase Ia is phosphorylated and is activated up to 50-fold. Autophosphorylation-autoactivation, if it occurs at all, does so at an extremely slow rate relative to phosphorylation and activation by the activator(30) , an observation consistent with the lack of significant autoactivation by bacterially expressed CaM kinase I(8) . Since CaM kinase Ia activator is able to phosphorylate and activate brain CaM kinase Ia inactivated by an irreversible ATP affinity analogue or a kinase-negative mutant of recombinant CaM kinase I, it may be concluded that the activator is itself a protein kinase, that is, a CaM kinase I kinase(31) .()In similar fashion to CaM kinase I, purified rat brain CaM kinase IV was reported to be subject to Ca-CaM-dependent autophosphorylation resulting in activation(32, 33) . Compared with the brain enzyme, recombinant CaM kinase IV expressed in bacterial or insect cells has high K values for substrates, slowly autophosphorylates, and demonstrates minimal activation(19, 34) . However, phosphorylation and activation of recombinant CaM kinase IV was found to be markedly stimulated by the addition of rat brain extract (35) or CaM kinase IV activators that appear themselves to be protein kinases(36, 37) .

We report here the phosphorylation-dependent activation of recombinant CaM kinase IV by purified CaM kinase Ia kinase. Thus, CaM kinases I and IV appear to be regulated by either common or highly related kinase kinases. We also report that replacement of Thr-196 of CaM kinase IV with Ala by site-directed mutagenesis prevents both phosphorylation and activation, indicating that Thr-196 phosphorylation is essential for activation.


EXPERIMENTAL PROCEDURES

Peptides

Syntide-2 was purchased from Life Technologies, Inc. GS10 was generously provided by Dr. Bruce Kemp (St. Vincent's Institute for Medical Research, Melbourne, Australia). Porcine and chicken calmodulin were obtained from Boehringer Mannheim or produced by bacterial expression(38) , respectively. PKI-tide (TTYADFIASGRTGRRNAIHD) was purchased from Sigma.

Expression and Mutagenesis of CaM Kinase IV

General molecular biology techniques were carried out according to Sambrook et al.(39) . CaM kinase IV was expressed in insect Sf9 cells using the baculovirus system as described previously(19) . Alternatively, CaM kinase IV was expressed in E. coli as follows. The cDNA encoding rat brain CaM kinase IV was subcloned as a pGCaMKIV (15) SacI/XbaI fragment into the SacI/XbaI sites of pGEX-3XMCS (pGEX-3X from Pharmacia Biotech Inc., modified so as to contain the multiple cloning site of pUC19), to generate pGEXCaMKIV. Recombinant protein was then expressed from pGEXCaMKIV in E. coli strain BL21 (DE3) as a glutathione S-transferase fusion protein and purified using glutathione-Sepharose. T196A mutant cDNA was constructed as follows. A 659-base pair cDNA fragment was prepared from a 30-cycle polymerase chain reaction using pGCaMKIV as template, an oligonucleotide primer (sense) overlapping a SacI site (5` CAC TAT AGG GCG AAT TCG AGC TCG GAC CCG GCG AAG ATG 3`), and, an oligonucleotide primer (antisense) overlapping a SmaI site that introduces the point mutation indicated by boldface type (5` AAT CTC AGG TGC ACA GTA CCC CGG GGT TCC ACA CAC CGC CTT CAT GAG 3`). The 659-base pair polymerase chain reaction product was subcloned as a SacI/SmaI fragment into the SacI/SmaI sites of pGCaMKIV to generate pGCaMKIVT196A. After confirming the presence of the mutated base and the correct ligation junctions by nucleotide sequence analysis using U.S. Biochemical Corp. Sequenase version 2.0 DNA sequencing kit and custom-designed oligonucleotides, the mutant cDNA was subcloned into pGEX-3XMCS and expressed and purified as described above.

CaM Kinase Ia Kinase

CaM kinase Ia kinase was purified from pig brain by a procedure previously described in detail(30) , modified by reversal of the order of the CaM-Sepharose and phenyl-Sepharose chromatographic steps and by omission of the hydroxylapatite chromatographic step.

Rat Brain Extract Preparation

Rat brain was homogenized with a Potter-Elvehjem homogenizer in 3 volumes of a buffer containing 5 mM Tris, pH 7.5 (at 4 °C), 0.1 mM DTT, 10 µg/ml antipain, 10 µg/ml leupeptin, 10 µg/ml phenylmethylsulfonyl fluoride, and 10 µg/ml trypsin inhibitor. After centrifugation of the homogenate at 100,000 g at 4 °C, the supernatant was desalted over Sephadex G-25 and stored in small aliquots at -70 °C.

Peptide Kinase Assay

The activity of CaM kinase IV was assayed at 30 °C using either Syntide-2 or GS10 as substrate as indicated in the figure legends. Syntide-2 kinase activity was measured by incubation in a mixture containing 50 mM Tris, pH 7.6, 1 mM CaCl, 1 µM CaM, 0.5 mM DTT, 0.5 mg/ml bovine serum albumin, 10 mM MgCl, 200 µM [-P]ATP (0.2 10 cpm/pmol), and 50 or 100 µM Syntide-2. GS10 kinase activity was measured by incubation in a mixture containing 50 mM Hepes, pH 7.5, 1 mM CaCl, 1 µM CaM, 30 µM PKI-tide, 10 mM MgCl, 50 or 200 µM [-P]ATP (0.1-1.2 10 cpm/pmol), and 40-50 µM GS10. Quantitation of P incorporation into peptides was determined by their binding to P81 phosphocellulose paper as described previously(5, 19) .

Phosphoamino Acid Analysis

P-labeled CaM kinase IV was subjected to SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto polyvinylidene difluoride membrane (Immobilon, Millipore) (100 V, 90 min). The membrane was then rinsed in deionized water, dried, and autoradiographed. Using the autoradiogram as template, the polyvinylidene difluoride strip containing P-labeled CaM kinase IV was excised from the blot, rinsed successively in methanol and water, and hydrolyzed in 6 N HCl (110 °C, 1 h) under vacuum. The P-labeled phosphoamino acids were dried, resuspended in deionized water containing phosphoamino acid standards, and subjected to two-dimensional separation by ascending chromatography in 2-propanol:HCl:HO (7:1.5:1.5) followed by high voltage electrophoresis (500 V, 140 min) in 7.5% acetic acid, 2.85% formic acid, pH 1.9. The standards and P-labeled phosphoamino acids were identified by ninhydrin staining and autoradiography, respectively. For relative quantitation of P incorporation, the cellulose corresponding to each phosphoamino acid was scraped from the plate using the autoradiogram as a template and counted in Ready-solve CP (Beckman) after extraction in methanol.

Other Methods

SDS-polyacrylamide gel electrophoresis was performed as described previously(19, 30) . The concentration of CaM kinase IV was determined by the method of Lowry et al. (40) as modified (5) or by the method of Bradford et al.(41) . CaM kinase Ia kinase concentration was determined by a colloidal gold staining method(30) . CaM was quantified by spectrophotometry (E = 3300).


RESULTS

The activity of recombinant CaM kinase IV is enhanced 24-fold to a specific activity of 1.7 µmol min mg by the presence of purified pig brain CaM kinase Ia kinase (Fig. 1). Unlike CaM kinase Ia, which remains fully Ca-CaM-dependent in the activated state(6, 30) , CaM kinase IV activation is accompanied by the development of significant (38%) Ca-CaM-independent activity. The time course and requirements for the activation of CaM kinase IV are illustrated in Fig. 2. As shown, the combined presence of CaM kinase Ia kinase, Ca-CaM, and MgATP is necessary for full activation to occur. The mechanism underlying the Ca-CaM-requirement for activator responsiveness, a phenomenon also observed for CaM kinase Ia(6, 30) , is under active investigation in our laboratories.


Figure 1: Activation of CaM kinase IV by CaM kinase Ia kinase (CaMKIa Kinase). Recombinant CaM kinase IV, expressed in Sf9 cells and purified as described under ``Experimental Procedures'' (1 µg/ml), was preincubated in the presence or absence of CaM kinase Ia kinase (0.1 µg/ml) as indicated for 120 min at 30 °C. All preincubations also contained 10 mM MgCl, 0.2 mM ATP, 1 mM CaCl, 1 µM CaM. Peptide kinase activity was then measured using Syntide-2 as substrate, as described under ``Experimental Procedures'' for 2 min with the exception that the kinase assay was performed in either the presence of 1 mM CaCl and 1 µM CaM or the absence of Ca-CaM and presence of 2 mM EGTA as indicated. Each bar represents the mean ± S.E. (n = 6). One unit of CaM kinase IV activity = 1 µmol of P transferred to Syntide-2 in 1 min.




Figure 2: Time course and requirements for CaM kinase IV activation. Recombinant CaM kinase IV, expressed in Sf9 cells and purified as described under ``Experimental Procedures'' (0.8 µg/ml), was preincubated in the presence (closedsymbols) or absence (opensymbols) of CaM kinase Ia kinase (0.1 µg/ml) for the indicated time periods of preincubation, at 30 °C, with the following additions (as final concentrations; CaCl and MgCl are given as concentrations in excess of EGTA and EDTA concentrations, respectively): 10 mM MgCl, 0.2 mM ATP (squares); 1 mM CaCl, 1 µM CaM (triangles); or 10 mM MgCl, 0.2 mM ATP, 1 mM CaCl, 1 µM CaM (circles). Peptide kinase activity was then measured using Syntide-2 as substrate in the presence of Ca (1 mM in excess of EGTA) and CaM (1 µM) as described under ``Experimental Procedures'' for 1-2 min. For background subtractions under each condition, activity in the absence of preincubation (zero time of preincubation) has been subtracted to control for activation occurring during the assay itself, and where appropriate, the activity of CaM kinase Ia kinase alone has also been subtracted. Each point represents the mean of duplicate determinations.



Based on the MgATP requirement for activation, we investigated whether CaM kinase IV is phosphorylated in the presence of CaM kinase Ia kinase. As shown in Fig. 3, CaM kinase IV is phosphorylated in a reaction requiring (in addition to MgATP), CaM kinase Ia kinase and Ca-CaM. The identical requirements for both activation and phosphorylation suggest that activation of CaM kinase IV is caused by its Ca-CaM-dependent phosphorylation by CaM kinase Ia kinase. Consistent with this mechanism is a linear relationship between the extent of phosphorylation and state of activation of CaM kinase IV (Fig. 4). The maximal extent of P incorporation achieved (6.5 ± 1.7 mol of P/mol of CaM kinase IV) demonstrated some variability between experiments but indicates multiple sites of phosphorylation. Phosphoserine is the predominant phosphoamino acid detected with a ratio of serine to threonine phosphorylation of 2.7 (± 0.6):1 (Fig. 5).


Figure 3: The phosphorylation of CaM kinase IV requires CaM kinase Ia kinase, Ca-CaM, and MgATP. Recombinant CaM kinase IV, expressed in Sf9 cells and purified as described under ``Experimental Procedures'' (7 µg/ml), and CaM kinase Ia kinase (0.5 µg/ml) were incubated alone or together for 15 min., at 30 °C, with the following additions as indicated (final concentrations; CaCl is given as concentration in excess of the EGTA concentration): 1 mM CaCl, 1 µM CaM; or 0.6 mM EGTA (CaM omitted). All incubations also contained the following: 50 mM Tris, pH 7.6, 0.5 mM DTT, 10 mM MgCl, and 20 µM [-P]ATP (3 10 cpm/pmol). Reactions were terminated by boiling in SDS--mercaptoethanol dissociation solution and electrophoresed in a 10% polyacrylamide SDS-gel. The resultant autoradiogram of the stained and destained gel is shown. The arrow indicates the position of CaM kinase IV.




Figure 4: Relationship between the phosphorylation and activation states of CaM kinase IV. Recombinant CaM kinase IV, expressed in Sf9 cells and purified as described under ``Experimental Procedures'' (2.8 or 4.9 µg/ml), and CaM kinase Ia kinase (0.5 µg/ml) were preincubated in a total volume of 76 µl at 30 °C, in a mixture containing 50 mM Tris, pH 7.6, 1 mM CaCl, 1 µM CaM, 0.5 mM DTT, 0.5 mg/ml bovine serum albumin, 10 mM MgCl, and 20 µM [-P]ATP (7 10 cpm/pmol). At five time points over a period of 2 h, replicate aliquots of the reaction mixture were separately and simultaneously analyzed for the states of activation and phosphorylation of CaM kinase IV. CaM kinase IV activation state was assessed by Syntide-2 kinase activity in the presence of Ca-CaM, utilizing background subtractions as described in the legend to Fig. 2. The phosphorylation of CaM kinase IV was determined by applying 8-µl aliquots of the preincubation reaction mixture to 2 2 cm squares of 3MM filter paper (Whatman). Squares were then washed once for 30 min at 0 °C in 10% trichloroacetic acid containing 2% sodium pyrophosphate followed by two washes for 15 min each in 5% trichloroacetic acid at 22 °C and dried with 95% ethanol. P-incorporation was quantified by liquid scintillation counting with subtraction of a CaM kinase Ia kinase alone condition as background. Results are expressed as the linear regression of activation versus phosphorylation after normalization to the maximal extent of both achieved. Each point represents the mean of duplicate determinations. Linear regression was performed using Sigmaplot version 1.02 (Jandel Scientific).




Figure 5: Phosphoamino acid analysis of phosphorylated CaM kinase IV. Recombinant CaM kinase IV, expressed in Sf9 cells and purified as described under ``Experimental Procedures'' (21 µg/ml), was incubated with CaM kinase Ia kinase (1 µg/ml) for 15 min., at 30 °C, in a mixture containing 25 mM Tris, pH 7.6, 1 mM CaCl, 1 µM CaM, 0.5 mM DTT, 10 mM MgCl, and 20 µM [-P]ATP (13 10 cpm/pmol). The reaction was terminated by boiling in SDS--mercaptoethanol dissociation solution and electrophoresed in a 10% SDS-polyacrylamide gel. P-labeled CaM kinase IV was then subjected to phosphoamino acid analysis by two-dimensional, ascending chromatography/high voltage electrophoresis as described under ``Experimental Procedures.'' The positions of phosphotyrosine (PY), phosphoserine (PS), and phosphothreonine (PT) are indicated.



Parallel studies in our laboratories have established that the phosphorylation of Thr-177 of recombinant CaM kinase I by CaM kinase Ia kinase is essential for CaM kinase I activation. In addition, Sugita et al.(42) reported that Thr-177 of CaM kinase V is a phosphorylation site for a CaM kinase kinase preparation. With this information as rationale, we addressed the critical question of the phosphorylation site(s) responsible for CaM kinase IV activation through site-directed mutagenesis of the equivalent amino acid residue in CaM kinase IV, Thr-196. As shown in Fig. 6and 7, replacement of Thr-196 with a nonphosphorylatable alanine abolishes both the phosphorylation and activation of CaM kinase IV by CaM kinase Ia kinase. Thr-196 phosphorylation is thus absolutely required for CaM kinase IV activation to occur.


Figure 6: Replacement of Thr-196 of CaM kinase IV with alanine prevents the phosphorylation of CaM kinase IV by CaM kinase Ia kinase. Recombinant CaM kinase IV wild type (CaMK IV wt) and T196A mutant, expressed in E. coli as glutathione S-transferase fusion proteins and purified as described under ``Experimental Procedures,'' were incubated (both at concentrations of 20 µg/ml) in the presence or absence of CaM kinase Ia kinase (0.04 µg/ml) as indicated for 10 min at 30 °C. All incubations also contained 50 mM Hepes, pH 7.5, 1 mM CaCl, 1 µM CaM, 30 µM PKI-tide, 10 mM MgCl, and 50 µM [-P]ATP (0.9 10 cpm/pmol). Reactions were terminated by boiling in SDS--mercaptoethanol dissociation solution and electrophoresed on a 10% polyacrylamide SDS-gel. The resultant autoradiogram of the stained and destained gel is shown. The arrow indicates the position of CaM kinase IV which electrophoreses with slower mobility than as illustrated in Fig. 3due to fusion with glutathione S-transferase.



Finally, despite the maximal extent of activation achievable (>20-fold, Fig. 1), there remained a possibility that additional components necessary for full activation are lost during CaM kinase Ia kinase purification. As shown in Fig. 8, although rat brain extract is capable of activating recombinant CaM kinase IV, this effect is no longer observed after activation by CaM kinase Ia kinase. These results indicate that purified CaM kinase Ia kinase is sufficient for full activation of CaM kinase IV.


Figure 8: Activity of CaM kinase IV (CaMKIV) in the presence and absence of CaM kinase Ia kinase (CaMKIa Kinase) and the presence and absence of rat brain extract. Recombinant CaM kinase IV, expressed in Sf9 cells and purified as described under ``Experimental Procedures'' (7.6 µg/ml), was preincubated in the presence or absence of CaM kinase Ia kinase (0.04 µg/ml) and in the presence or absence of rat brain extract (15 µg/ml) as indicated for 30 min at 30 °C. All preincubations also contained 50 mM Hepes, pH 7.5, 1 mM CaCl, 1 µM CaM, 10 mM MgCl, and 200 µM ATP. Peptide kinase activity was then measured using GS10 as substrate, as described under ``Experimental Procedures'' for 6 min. Each bar represents the mean ± S.E. (n = 2) with the exception that the activity of the brain extract alone was calculated as the difference between CaM kinase Ia kinase plus brain extract and CaM kinase Ia kinase alone.




DISCUSSION

In a number of respects, the responses of CaM kinases Ia and IV to CaM kinase Ia kinase are virtually identical. Both kinases are maximally activated >20-fold in reactions requiring CaM kinase Ia kinase, Ca-CaM, and MgATP, and both are phosphorylated with these same requirements. Nonetheless, we have observed several prominent differences between the two CaM kinases. The first is that while the activity of CaM kinase Ia is completely Ca-CaM-dependent after activation(6, 30) , CaM kinase IV develops significant (38%) Ca-CaM-independent activity as a result of activation. Second, while incubation of CaM kinase Ia with CaM kinase Ia kinase in the presence of Ca-CaM and MgATP results in phosphorylation almost exclusively on threonine(5, 31) , incubation of CaM kinase IV under the same conditions leads to the phosphorylation of multiple serine and threonine residues with a Ser-P/Thr-P ratio of 3:1 (Fig. 5). These observations are similar to those of McDonald et al.(43) who reported multiple CaM kinase IV autophosphorylation sites with serine the predominant phosphoamino acid detected. Since there is no significant phosphorylation of the T196A mutant enzyme (Fig. 6), most of the observed P-incorporation appears to be generated in a burst of secondary autophosphorylation reflecting the activated state of CaM kinase IV. In this scenario, the close temporal relationship between total CaM kinase IV phosphorylation and activation, evident from the linear relationship between the two (Fig. 4), implies that autophosphorylation rapidly follows Thr-196 phosphorylation and that the former is therefore, in all probability, intramolecular. Supportive of this hypothesis is the observation that a kinase-negative mutant of CaM kinase IV is phosphorylated in the presence of CaM kinase Ia kinase to a considerably lesser extent than is the wild type enzyme.()

Whatever its mechanism, the presence of multiple phosphorylation sites raises a question as to whether phosphorylation of an amino acid residue other than Thr-196 is ultimately responsible for induction of the Ca-CaM-independent activity of CaM kinase IV. That this indeed may be the case is suggested by two additional observations. First, as noted above, CaM kinase I does not acquire Ca-CaM independence after phosphorylation by CaM kinase Ia kinase (6, 30) even though phosphorylation occurs at a site (Thr-177) equivalent to Thr-196 in CaM kinase IV. Activation and Ca-CaM independence are therefore, for any given case, potentially dissociable phenomena. Second, precedence for the generation of a Ca-CaM-independent CaM kinase activity was established through studies of the autophosphorylation of CaM kinase II. In this case, phosphorylation occurs in the autoinhibitory domain itself (Thr-286 in CaM kinase II, (44, 45, 46, 47) ), whereas in CaM kinase IV, Thr-196 is considerably removed, at least in the linear sequence from the autoinhibitory domain, identified as including residues 313-323 (19) or 308-317(36) . Thus, apart from the activation process per se, elucidation of the mechanism of generation of Ca-CaM-independent CaM kinase IV activity may yield insights into the means for prolongation of the duration of action of Ca-mobilizing signals.

The complete inability of the T196A mutant to be both phosphorylated and activated provides direct evidence that phosphorylation of Thr-196 is essential for the activation of CaM kinase IV ( Fig. 6and Fig. 7). Nevertheless, it is conceivable that the T196A substitution by itself could induce a conformational change that blocks activation. The latter possibility would, however, appear to be highly unlikely considering that this is a single conservative replacement and that in all of its properties we have tested other than its inability to be activated the T196A mutant enzyme is indistinguishable from the wild type enzyme. For example, its basal specific activity is the same as that of the wild type enzyme (Fig. 7); it binds calmodulin in a CaM overlay procedure; and it is of equivalent immunoreactivity as the wild type enzyme using a CaM kinase IV antibody. It should also be noted that the requirement for Thr-196 phosphorylation does not preclude the possibility that this event may induce phosphorylation at other sites that may also participate in the activation process.


Figure 7: Replacement of Thr-196 of CaM kinase IV with alanine prevents the activation of CaM kinase IV by CaM kinase Ia kinase (CaMKIa Kinase). Recombinant CaM kinase IV wild type (CaMKIVwt) and T196A mutant, expressed in E. coli as glutathione S-transferase fusion proteins and purified as described under ``Experimental Procedures,'' were preincubated (both at concentrations of 16 µg/ml) in the presence or absence of CaM kinase Ia kinase (0.04 µg/ml) as indicated for 10 min at 30 °C. All preincubations also contained 50 mM Hepes, pH 7.5, 1 mM CaCl, 1 µM CaM, 10 mM MgCl, and 50 µM ATP. Peptide kinase activity was then measured using GS10 as substrate as described under ``Experimental Procedures'' for 6 min. Each bar represents the mean ± S.E. (n = 2).



Thr-196 is found in the sequence LMKTVCGTPGYCAPE(15) , contained in the catalytic domain in subdomain VIII, a region highly conserved among protein kinases(48) . It has recently become apparent that for many protein kinases, phosphorylation of a Ser or Thr residue located 9 or 10 residues amino-terminal of the nearly invariant (A/S)PE sequence is essential for the expression of full catalytic activity. Kinases for which this phenomenon has been demonstrated include the following: cAMP-dependent protein kinase catalytic subunit (49) , protein kinase C(50) , mitogen-activated protein kinase(51) , mitogen-activated protein kinase kinase-1 (52, 53) and the cyclin-dependent kinases, cdc 2, cdk 2, and cdk 4(54, 55, 56) . This list has now been expanded to include the CaM kinase family members, CaM kinase IV (this report), and CaM kinase I.

The prevalence of these observations raises a question as to the ultimate physiological importance of such phosphorylation events. In some instances, for example in that of cAMP-dependent protein kinase, this phosphorylation may be autocatalytic following translation and resistant to intracellular phosphatase action(49, 57) . There are, however, a number of lines of evidence to suggest that in some cases, these phosphorylations represent a form of acute regulation and serve cell-specific functions. First, although a requirement for a phosphorylated residue (or acidic residue as a phosphoamino acid mimetic) at this position may be common, it is by no means universal. For example, within the CaM kinase family such residues are absent from CaM kinase II (58, 59, 60) and myosin light chain kinase isoforms(61, 62) . Second, the ability to isolate CaM kinase Ia from tissue in the nonactivated, i.e. dephosphorylated state, and the ability to reverse activation with purified phosphoprotein phosphatase 2A (6) imply that dephosphorylation is reasonably facile and that there is, therefore, the potential for a phosphorylation/dephosphorylation cycle in vivo. Third, although perhaps autocatalytic in the case of cAMP-dependent protein kinase, it is now established that in most of the other cases, cited above, a heterologous kinase(s) is involved, and in some of the other cases, for example mitogen-activated protein kinase, the signal transduction pathway involved has been elucidated.

Two recent reports (36, 37) described 66-68-kDa CaM kinase IV activators from rat brain that appear themselves to be protein kinases. And another report described a preparation capable of activating and phosphorylating both CaM kinase I and CaM kinase IV(42) . It will ultimately be of interest to determine the relationship between these CaM kinase activators and the 52-kDa CaM kinase Ia kinase purified from pig brain(30) , which was used here. Moreover, as noted previously, CaM kinase Ia kinase activity demonstrates chromatographic heterogeneity during purification, suggesting the existence of isoforms (30) . Thus, although, CaM kinase Ia kinase is capable of fully activating CaM kinase IV (Fig. 8), it is possible that there are multiple CaM kinase I/CaM kinase IV activating kinases and that these enzymes have similar substrate specificities but relative preferences for the two CaM kinase targets. It is also possible, however, that there are species differences between rat and pig brain CaM kinase activators, which may account for the apparent differences in reported molecular weights. A final possibility is that there are distinct activators that produce their effects by different mechanisms. For example, it was proposed that phosphorylation of Ser-437 results in CaM kinase IV activation (33) and that CaM kinase IV is phosphorylated by the 66-kDa rat brain CaM kinase IV activator kinase exclusively on serine residues(37) . Also Tokumitsu et al. (36) have hypothesized that a 68-kDa CaM kinase IV activator kinase may recognize argininyl residues amino-terminal of its phosphorylation site. It will be of importance, therefore, to evaluate these suggestions in light of the information presented here indicating that Thr-196 phosphorylation is essential for CaM kinase IV activation.

The upstream regulatory signals that may serve to drive CaM kinase kinase-catalyzed phosphorylation of CaM kinases I and IV is an area of active investigation in our laboratories. An example of the form this regulation might take was suggested by Hanissian et al.(63) , who observed that in Jurkat cells, in addition to Ca-influx, a signal delivered through TCR.CD3 is required for full CaM kinase IV activation, an observation consistent with a need for an additional signal for the activation of CaM kinase IV kinase(s). Finally, it may be noted that whatever the precise physiological role of these phosphorylation events for the CaM kinase family, the existence of CaM kinase kinase(s) as well as the earlier emergence of the concept of Ca-CaM-independent CaM kinase activity, suggest that a full appreciation of the complexity of regulation of these key signal-transducing enzymes has yet to be attained.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants NS24738 (to A. M. E.), GM07145-20 (training grant support for M. A. S.), and HD07503 and GM33976 (to A. R. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 716-829-3491; Fax: 716-829-2801; aedelman{at}ubmede.buffalo.edu

The abbreviations used are: CaM, calmodulin; CaM kinase, Ca-CaM-dependent protein kinase; Syntide-2 and GS10, synthetic peptides PLARTLSVAGLPGKK and PLRRTLSVAA, respectively, based on phosphorylation site 2 in glycogen synthase; PKI-tide, synthetic peptide based on the heat-stable inhibitor of cAMP-dependent protein kinase (amino acid residues 5-24); DTT, dithiothreitol.

Haribabu, B., Hook, S. S., Selbert, M. A., Goldstein, E. G., Tomhave, E. D., Edelman, A. M., Snyderman, R., and Means, A. R. (1995) EMBO J., in press.

The terms CaM kinase Ia activator, CaM kinase Ia kinase, and CaM kinase I kinase are used synonymously.

K. Anderson and A. R. Means, unpublished observations.


ACKNOWLEDGEMENTS

We are indebted to Dr. J. C. Lee for performing valuable preliminary studies. We also thank Dr. J. Aletta for helpful discussions and T. Atkinson for photographic illustrations.


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