C The Ca -Calmodulin Dependent Protein Kinase II System of Skeletal Muscle Sarcoplasmic Reticulum

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Skeletal Muscle Sarcoplasmic Reticulum

Roberta Sacchetto, Elisa Bovo and Ernesto Damiani

Dipartimento di Scienze biomediche sperimentali, Università di Padova, Padova

Abstract

It is well known that skeletal muscle sarcoplasmic reticulum (SR) contains a Ca²⁺- calmodulin (CaM) dependent protein kinase (CaMKII) system. In the last twenty years, much experimental work has clarified the biochemical-functional properties of this kinase system. SR-bound CaMKII is formed by the holoenzyme and by anchoring proteins, called αKAP. The holoenzyme is composed by a muscle specific form of β subunit (β_Μ), and by γ and δ subunits. In rabbit skeletal muscle, β_Μ seems to be expressed in greater amounts in slow-twitch muscle. Two isoforms of αKAP of 23-21 kDa are present in isolated SR fragments of rabbit fast-twitch muscle, whereas only the 23 kDa αKAP splice variant is expressed in slow-twitch muscle. Based on biochemical work on isolated SR vesicles, the kinetic properties of SR-bound CaMKII do not significantly differ from those of CaMKII in other tissues. CaMKII is localized on the cytoplasmic side of SR membranes, and is ubiquitously distributed between longitudinal SR and junctional SR. In slow-twitch muscle, CaMKII is mainly involved in regulation of SR Ca²⁺-transport, as indicated by phosphorylation of SERCA2a isoform of Ca²⁺-ATPase and of SERCA2a-regulatory protein phospholamban. In fast-twitch muscle, the main role of CaMKII seems to be the control of Ca²⁺- release. Much experimental evidence seems to negate that ryanodine receptor/SR Ca²⁺- release channel of skeletal muscle (RyR1) is a substrate of phosphorylation of endogenous CaMKII. The main undisputed, junctional SR-specific protein substrate of CaMKII is triadin, which interacts structurally and functionally with RyR1. The functional effect on RyR1 of CaMKII-dependent phosphorylation of junctional SR membranes remains somewhat controversial, even though the bulk of experimental evidence suggests a negative regulation of the calcium channel by the kinase. Additional protein substrates of SR-bound CaMKII are histidine-rich Ca²⁺-binding protein, glycogen synthase and 21 kDa αKAP.

Therefore, SR-bound CaMKII is involved in regulation of Ca²⁺-homeostasis and, likely, of glucose metabolism. A putative role in regulation of gene expression remains to be established.

Key words: αKAP, Ca²⁺-calmodulin dependent protein kinase, Ca²⁺ homeostasis, Ca²⁺- release channel/ryanodine receptor, sarcoplasmic reticulum, skeletal isoform, skeletal muscle, triadin.

Basic Appl Myol 15 (1): 5-17, 2005

C

a²⁺-calmodulin (CaM) dependent protein kinase II [CaMKII] is a ubiquitous, pleiotropic serine/threonine protein kinase. Upon elevation of intracellular Ca²⁺ concentration, apo-CaM binds Ca²⁺. In turn, Ca²⁺-CaM binds to CaMKII, which becomes activated and able to act on a broad spectrum of substrates in vitro and in vivo. For this reason, CaMKII is called multifunctional.

This wide substrate specificity allows CaMKII to play a role in the regulation of a multiplicity of physiological processes (see ref. [42, 69, 72], for recent reviews).

CaMKII holoenzyme is a heteromultimer, containing between 6 and 12 different subunits [4, 5, 41, 69, 72]. It

is, however, known that homomultimers may exist [5].

Each subunit is the product of one out of four distinct genes, called α, β, γ and δ. The subunits range in size from 54 to 72 kDa. To date, several isoforms have been identified for each subunit [3, 5, 81]. This is an important point, since differences in subunit composition may modify the functional properties of the holoenzyme [5, 6; 27]. α and β CaMKII isoforms are predominantly [5, 72], or almost exclusively [56, 4] expressed in neural tissues, whereas γ and δ isoforms are more widely expressed in non-neuronal tissues [5, 56, 72].

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The core structures of the different CaMKII isoforms are identical, each subunit being formed by three domains, i. e. the N-terminal catalytic domain, a central regulatory domain and a C-terminal association domain.

The central domain contains the regulatory CaM- binding region, as well as the autoinhibitory region (AID). A conserved linker region is inserted between the catalytic and the regulatory domains [43, 69]. All isoforms display a high degree of structural homology (80-90%) in the N-terminal catalytic and central regulatory domains, the most variable region being the C- terminal association domain [5]. Inserts numbered from I to XI [see 69, 43, for nomenclature] are introduced into one or more of four variable regions, giving raise to multiple splice variants. Todate, up to 30 isoforms of CaMKII have been described [43]. However, in general terms, the CaMKII isoforms display similar, even though not identical, catalytic and regulatory properties [4, 27].

The association domain is necessary for the mul- timerization of the holoenzyme. In fact, the association domain of each subunit binds to the association domain of adjacent subunits, forming the central core of the holoenzyme, whereas the N-terminal catalytic domains project outward. Therefore, the holoenzyme is arranged in a hub-and-spoke pattern [5], forming a wheel-like pattern (see also [27], [54]).

The hub-and-spoke pattern is essential to understand the regulative properties of the holoenzyme. In the presence of apo-CaM, i. e. at low [Ca²⁺]i, the AID interacts intramolecularly with its own catalytic domain, thereby inhibiting the catalytic activity. When Ca²⁺ binds to CaM, Ca²⁺-CaM complex interacts with the N-terminal region of the AID [5], causing a conformational change, which disrupts the interaction between the AID and the catalytic domain, thereby activating the enzyme. At this point, each subunit undergoes autophosphorylation at Thr-286 of α CaMKII isoform (Thr-287 of the other subunits), becoming able to phosphorylate the same site of the proxymate subunits within the same holoenzyme [5]. It is, therefore, an intramolecular, intersubunit- catalyzed phosphorylation process [5, 57, 72], in which each subunit acts, in turn, as a substrate and as a kinase.

The autophosphorylation of Thr-286 has two important consequences: a) it increases the affinity of CaMKII for CaM from the nM to the pM range [5, 72]; b) it makes CaMKII independent of Ca²⁺ and calmodulin. In the Ca²⁺-CaM independent form, CaMKII undergoes additional autophosphorylation at Thr-305 or Thr-306 [25]. This unique regulatory mechanism allows the enzyme to phosphorylate independently of Ca²⁺-CaM its substrates, even when the intracellular Ca²⁺ concentration has returned to basal, sub-µM level. These unique autoregulative features of CaMKII make the kinase sensitive to the frequency, duration and amplitude of intracellular Ca²⁺ variations [21].

The CaMKII consensus sequence for substrate recog- nition is R/K-X-X-S/T [5, 61, 69].

CaMKII is highly enriched in the nervous system [25, 69], its level in non-neural tissues being about 1/50 that of brain, including skeletal muscle [69]. In the nervous system, a variable proportion of the enzyme is soluble in the cytoplasm [25], but most of CaMKII is particulate and restricted to subcellular pools, such as postsynaptic densities [56, 72]. For this reason, it is generally as- sumed that CaMKII might play a role in the mechanism of learning and memory [5, 43]. Furthermore, some splice variants of α (αΒ), δ (δΒ) and γ (γΑ) isoforms contains a Nuclear Localization Sequence (NLS), inserted into the variable region following the regulatory domain [35, 73]. This motif, KKRK, allows the kinase to be targeted to the nuclei [35, 73] of neuronal cells [79], as well as of ventricular myocytes [62, 92].

Three Pools of Ca²⁺/Calmodulin-Dependent Pro- tein Kinase Exist in Skeletal Muscle

As for other tissues (see Fig. 13. 1 of [69]), skeletal muscle contains three pools of CaMKII, i. e. nuclear, cytosolic and membrane-bound.

Concerning nuclear localization, Fluck et al. [26] reported that CaMKII enriches in nuclei extracts of skeletal muscle, and phosphorylates the nuclear transcription factor SRF in vitro. The Authors suggested that SRF phosphorylation may directly influence its binding to DNA. This observation suggest that Ca²⁺ might regulate gene transcription not only through a Ca²⁺/CaM/calcineurin pathway, but also through a Ca²⁺/CaM/CaMKII pathway. To the best of our knowledge, however, this report stands solitarily in this claim.

Furthermore, the relation of this CaMKII to cytosolic and to SR-bound CaMKII is still obscure.

Very little is known concerning cytosolic CaM- dependent protein kinase. Sato et al. [68] succeded in purifying the enzyme from the cytosol of rabbit hind- legs skeletal muscle, by CaM-affinity chromatography.

The purified enzyme migrated as a main protein band of 58 kDa and a minor band of 55 kDa. Both proteins, however, displayed CaM binding ability and cross- reacted with polyclonal antibodies to brain CaMKII, suggesting that they corresponded to two different CaMKII subunits. The cytosolic form of CaMKII was found to differ from SR-bound CaMKII, since the latter could be solubilized only by detergents [49]. In retro- spect, this result was likely due to the fact that cytosolic CaMKII does not contain any anchoring proteins [19], which are responsible for binding of CaMKII to SR membranes [2].

The analysis of partition of CaMKII holoenzyme between the cytoplasm and SR membranes, suggests that in rabbit fast-twitch skeletal muscle, CaMKII is predominantly bound to SR, whereas in slow-twitch muscle, a larger proportion of CaMKII is recovered in the cytoplasm (Figure 1).

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As far as the problem of possible differences in subunit composition between the cytoplasmic and SR- bound CaMKII, Figure 1 shows that the subunit composition of the holoenzyme similar between the cytosolic and SR-bound CaMKII. Identical results were obtained, when CaMKII was purified by affinity chromatography from soluble cytoplasm and SR membranes [E.

Damiani, unpublished observations]. At the present time, there is no evidence whatsoever suggesting a possible translocation of the holoenzyme form the cytosol to SR, and viceversa.

The Ca²⁺/Calmodulin-Dependent Protein Kinase of Skeletal Muscle Sarcoplasmic Reticulum

The knowledge that skeletal muscle SR contains a CaM-dependent protein kinase system goes back to the early eighties. At that time, the research on the possible association of a CaMKII system to the SR of skeletal muscle was prompted by the demonstration that, in cardiac muscle, the SR Ca²⁺-ATPase (SERCA2) was regulated by CaM-mediated phosphorylation of the regulatory protein phospholamban [47, 51].

Almost contemporarily, Chiesi and Carafoli [10] and Campbell and MacLennan [8] reported the existence of Ca²⁺-CaM-dependent protein kinase activity in the isolated SR of rabbit fast-twitch skeletal muscle. Chiesi and Carafoli [10, 11] described CaM-dependent phosphorylation of proteins of 57, 35 and 20 kDa, whereas

Campbell and MacLennan [8] and MacLennan et al.

[53] reported CaM-dependent phosphorylation of three proteins of 85, 60 and 20 kDa. Later, using CaM- sepharose affinity-chromatography, Tuana and MacLennan [83] purified CaMKII from isolated SR membranes of rabbit fast-twitch muscle. The purified CaMKII complex consisted of 89, 60, 34 and 20 kDa phosphoproteins, the 60 kDa polypeptide being the major CaM-binding protein. It has since become firmly established that the 57-60kDa phosphoprotein and the CaM-binding protein described in these initial studies correspond to endogenous CaMKII [12, 15, 49], as de- finitive demonstrated in immunoblots using specific antibodies raised against the Thr-286-phosphorylated form of CaMKII [19].

In their studies, both Chiesi and Carafoli [10] and Campbell and MacLennan [8] found that phosphorylation of SR proteins was triggered by addition of Ca²⁺ to the assay medium, even in the absence of CaM. Using indirect methods, Chiesi and Carafoli [10] suggested that isolated SR membranes contained up to 0.5-1 µg of CaM per mg of SR protein. These results were later confirmed by Eibschutz et al. [23], using microsomal preparations enriched in SR membranes from rabbit fast and slow muscle. These results obtained with isolated SR membranes, were in strong agreement with those obtained by using indirect immunofluorescence by Harper et al. [32], who found that, in skeletal muscle, CaM localized to the entire I band. The staining of the I band was reduced, but not completely abolished, after α-amylase treatment, which digested glycogen and, therefore, released glycogen-associated phosphorylase kinase, whose δ subunit is CaM [7]. Taken together, these results strongly suggested that a discrete pool of CaM was bound to SR membrane protein receptors.

More recently, Sacchetto et al. [66] used rabbit adduc- tor muscle, a representative fast-twitch muscle, to show that CaM displays a pattern of immunofluorescence typical of proteins localising to the triadic junction, i. e.

paired rows of fluorescent dots at the I band, without immunostaining of the Z line. These observations are consistent with the current knowledge that several high- affinity, CaM-binding proteins are present in the SR and in transverse tubules (TT), the major being CaMKII it- self, the SR ryanodine receptor/Ca²⁺-release channel (RyR1) [85], and the TT L-type, dihydropyridine sensitive Ca²⁺-channel [31].

Finally, using a two-dimensional SDS-PAGE system (EGTA in the first dimension, Ca²⁺ in the second dimension) followed by Stains all-staining, Damiani et al.

[16] identified CaM protein in SDS-polyacrylamide gels of highly-purified terminal cisternae (TC) isolated from rabbit fast muscle. The amount of CaM bound to TC was estimated to be approximately 50 pmol/mg of TC protein. This finding was in agreement with values pre- viously reported for isolated heavy SR vesicles from pig skeletal muscle (30 pmol/mg SR protein, see [91]).

Fast muscle Slow muscle

Cytosol

SR SR

βM

CaMKII

γ 75

50 kDa 100

Figure 1. Comparison between rabbit fast and slow muscle, regarding the partition of CaMKII be- tween cytosol and SR membranes. Muscle sub- fractions were isolated by the method of Saito et al. [1984]. Aliquots [50 µg] of soluble cyto- plasmic fraction and of isolated SR membranes were electrophoresed by 10-15% SDS-PAGE.

Proteins were transferred onto nitrocellulose, and blots were probed with a polyclonal anti- body to γ CaMKII, known to cross-react with βM

subunit [Damiani et al., 2000]. Only the molecu- lar weight region between 100 kda and 50 kDa is shown. The position of individual CaMKII subunits is indicated. The several isoforms of γ subunit are localized to 60 kDa, and the β_M sub- unit to 72 kDa. Identical results were obtained, using a goat polyclonal antibody to δ subunit.

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Taken together, these results clearly indicate that a sub- stantial pool of apo-CaM is tethered to SR membranes in close proximity of the RyR1 channels responsible for activator Ca²⁺ and of the RyR1’s effector, CaMKII.

Characterization of kinetic properties of SR-bound CaMKII

The kinetic parameters of SR-bound CaMKII have been analyzed in depth. Using low ATP concentrations (10 µM), Campbell and MacLennan [8] reported maximal autophosphorylation of CaMKII after 90 sec. Using 200 µM ATP, Chiesi and Carafoli [11] reported much rapid autophosphorylation of 60 kDa CaMKII (t1/2= 5- 10 sec at 0^o C). Phosphorylation experiments performed with TC isolated from rabbit skeletal muscle, and carried out at saturating concentrations of ATP (400 µM), Ca²⁺ (10 µM) and CaM (6 µM), later confirmed that half-maximal autophosphorylation of 60 kDa CaMKII was achieved in less than 30 sec [15, 19, see also Figure 2, A]. In Figure 2, B, the kinetics of autophosphorylation was monitored, using phosphorylation site-specific antibodies raised against P-Thr²⁸⁶ of CaMKII. It is clear that, in the presence of optimal experimental conditions, autophosphorylation is a very rapid reaction, reaching maximal level between 30-60 sec.

Concerning the maximal phosphorylation of SR proteins, all studies confirmed that Ca²⁺-CaM-dependent phosphorylation of substrates followed autophosphorylation of CaMKII [8, 10, 11, 15, 53], even though some substrates were phosphorylated more rapidly than others [19].

As far as the affinity for CaM of SR-bound CaMKII is concerned, Campbell and MacLennan [8] initially reported that half-maximal rate of phosphorylation was reached with 0.1 µM CaM. Chiesi and Carafoli [11]

later reported a much higher Km for CaM (1-2 µM).

However, the discrepancy between the two reports is likely due to the low free Ca²⁺ concentration (0.7 µM) used by Chiesi and Carafoli [11] in their phosphorylation experiments. In fact, kinetic parameters of phosphorylation are dependent on the reaction conditions [27]. Phosphoprylation experiments carried out on isolated TC from rabbit skeletal muscle at 100 µM free Ca²⁺ and at 400 µM ATP, indicated that about 0.05–0.1 µM CaM was required for half-maximal autophosphorylation of 60 kDa CaMKII [16, 19, 13, see also Figure 3, B]. At the same free [Ca²⁺] in the assay medium, Pelosi and Donella-Deana [60] reported that half- maximal activity of CaMKII was attained at about 0.3 µM CaM. These values are very similar to K_0.5 values reported by Gaertner et al. [27] for CaM-dependence of autophosphorylation of the different CaMKII isoforms.

Finally, the affinity for CaM of SR-bound CaMKII was roughly estimated by Damiani et al. [16], using a ligand-blot overlay technique with derivatized CaM.

Blotted CaMKII was found to bind CaM with high affinity, i. e. about 0.01 µM, at 100 µM free Ca²⁺. This

value was well in the range of values reported by Gaert- ner et al. [27] for each isoform of CaMKII.

Taken together, these results indicate that, at high Ca²⁺

concentrations, the CaM-binding and catalytic properties of SR-bound CaMKII are similar to those of individual CaMKII isoforms, and at least comparable to those of CaMKII of brain [5, 56] or of gastric mucosa [24].

In phosphorylation experiment carried out at 2 µM CaM and 0.7 µM free Ca²⁺, i. e. suboptimal concentrations of Ca²⁺, Chiesi and Carafoli [11] obtained a Km for ATP of about 200 µM. On the other hand, Campbell and MacLennan [8] found that the maximal autophosphorylation of CaMKII occurred with 50 µM ATP, without however specifying the concentrations of free Ca²⁺ and CaM used in their experiments. Using optimal concentrations of Ca²⁺ (10 µM) and CaM (5 µM) Pelosi and Donella-Deana [60] reported a Km for ATP of 23 µM. From these results, it is clear that the affinity for

Time (sec) 30 60 90 120 180 240 300 High Mr

HRC

triadin 60 kDa CaMKII

A

0 60 120 300 600 Time (sec) 30

Autoradiography

B

P^Thr286-CaMKII

Immunoblot

Figure 2. Kinetic of autophosphorylation of CaMKII.

[A] Phosphorylation of TC vesicles isolated from rabbit fast-twitch muscle by the method of Saito et al., [1984] was carried out at 0^o C with 400 µM [γ-³²P]ATP, at pCa 4 and in the pres- ence of 6 µM calmodulin for the indicated times.

32P-labelled phosphpproteins were detected by auroradiography. The position of individual pro- teins was indicated on the left. Key to abbrevia- tions: High Mr: High molecular weight protein;

HRC: histidine-rich, Ca²⁺-binding protein; 60 kDa CaMKII: the overall autophosphorylated material at 60 kDa, comprising γ and δ subunits of CaMKII. [B] TC vesicles phosphorylated as described in panel A for the indicated times.

Protein aliquots were electrophoresed and blot- ted. Blots were incubated with phosphorylation site-specific [P-Thr²⁸⁶] antibodies to autophos- phorylated CaMKII.

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ATP of SR-bound CaMKII is comparable to that observed for other calmodulin-dependent kinases [86].

Concerning the Ca²⁺-dependence of phosphorylation, using saturating concentrations of ATP (200 µM) and CaM (1.5 µM) in their phosphorylation assay, Chiesi and Carafoli [11] reported a K0.5 value for [Ca²⁺] of 0.7- 1.0 µM, which is in close agreement with results shown in Figure 3, B. Campbell and MacLennan [8] reported a somewhat lower value (0.3 µM Ca²⁺) for maximal phosphorylation in the presence of 0.6 µM CaM. Using ligand blot techniques and digoxigeninated CaM, Damiani et al. [16] found that half-maximal binding of CaM to blotted 60 kDa CaMKII occurred at around 1 µM Ca²⁺. Finally, Pelosi and Donella-Deana [60] reported a K0.5 for Ca²⁺ of 4.3 µM, at saturating CaM concentrations. In sum, these results indicate that the affinity of the enzyme for Ca²⁺ is comparable to that observed for CaMKII purified from other tissues (K0.5 for Ca²⁺ = 2.7 µM for CaMKII from gastric mucosa, see ref. [24]).

Subunit composition of SR-bound CaMKII system It is now well established that the SR-bound, multifunctional CaMKII system of skeletal muscle is composed by the heteromeric holoenzyme and by an anchoring protein.

Full-length subunit composition of the holoenzyme Using RNA blot analysis, Tobimatsu and Fujisawa [81] first reported that β, γ, and δ subunits of CaMKII, but not α subunit, were expressed in rat skeletal muscle.

These results were later confirmed by Bayer et al. [3], for skeletal muscle of adult mice. The skeletal muscle β subunit was found to be a muscle-specific isoform of 72 kDa, called termed β_M [2, 3]. At the protein level, Bayer et al. [2] examined by immunoblot analysis the CaMKII isoform composition of rat skeletal muscle microsomes, without however specifying the type of muscle used.

Based on their mobility in SDS-PAGE, CaMKII isoforms detected were 72 kDa β_M, γ_B and δ₄ (or δ_D). Again, α CaMKII full length subunit was not detected.

It is now known that there exist at least thirteen isoforms of δ CaMKII [43]. Using PCR analysis of total mRNAs from rat psoas muscle (a predominantly fast- twitch muscle), Schworer et al. [70] identified δ₄ as the main δ isoform expressed. By semiquantitative RT- PCR, Hagemann et al. [29] confirmed that δ4 was specifically expressed in skeletal muscle of adult rats, in addition to δ₂ and δ₉ isoforms. On the other hand, using a similar RT-PCR technique, Hoch et al. [37] were able to demonstrate that the transcripts for δ₂ and δ₉ were expressed in human thigh skeletal muscle, in addition to a skeletal-muscle specific, new isoform, termed δ₁₁. These results, therefore, suggest the existence of species-specific differences between small rodents and man, concerning the pattern of expression of δ CaMKII isoforms. It is not known whether different δ isoforms localize to different SR compartments.

As far as rabbit skeletal muscle is concerned, it is now clear that β_M, γ and δ isoforms are expressed both in fast- twitch [13, 17, 19, 67] and slow-twitch [18, 65] muscles.

However, the ratio between βM,and γ and δ subunits seems to be fiber-type specific, being higher in slow- twitch than in fast-twitch muscle [17, 19, 65]. At the present time, it is not known, if and how these differences in subunit composition may impact on the functional properties of the holoenzyme. It is, however, important to say that recently, Gaertner et al. [27] carried out an in-depth comparative characterization study of enzymatic properties of CaMKII isoforms, showing significant differences in their interactions with Ca²⁺-CaM, substrate phosphorylation and autophosphorylation.

Finally, β_M, γ and δ isoforms of CaMKII were detected also in human skeletal muscle [64].

Anchoring protein

Almost contemporaneously, Bayer et al. [1] and Sugai et al. [75] reported the existence in skeletal muscle of a CaMKII anchoring protein, which was responsible for targeting of the holoenzyme to SR. This was the first example of an anchoring protein for Ca²⁺-CaM- dependent protein kinases.

Using a probe which contained the 3’-untranslated region of α CaMKII, and Northern blot techniques, Bayer et al. [1] first found in mouse skeletal muscle a single, α CaMKII-related transcript, which corresponded to the mRNA for an alternative, non-kinase product of α CaMKII gene. Identical results were obtained for rat skeletal muscle by Sugai et al. [75]. The anchoring protein was termed αKAP (αCaMKII Association Protein) [1]. The C-terminal region of αKAP was identical to the association domain of α CaMKII gene, with the excep- tion of an 11 aminoacid insert in the variable region. On the contrary, the N-terminal domain of αKAP is not present in any known CaMKII protein, and is highly hydrophobic. Following the nomenclature of domains

High M_r

HRC

triadin 60 kDa CaMKII

CaM

( M)µ 0.01 0.05 0.1 0.5 1.0 2.0 6.0 pCa 7 6 5 4 3

A B

Figure 3. Functional characterization of SR-bound CaMKII from rabbit skeletal muscle. TC vesicles were phosphorylated at at 0^o C with 400 µM [γ-

32P]ATP. [A] The dose-dependence of CaM stimulation of protein phosphorylation was in- vestigated at pCa 4. [B] The pCa dependence of protein phosphorylation was analyzed in the presence of 2 µM calmodulin. Abbreviations are as in Figure 2, A.

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and inserts of Schulman and Braun [69] and of Hudmon and Schulman [43], the linear structure of αKAP is I-B- III-C-D, where I and III are inserts introduced into variable regions V1 and V2, B is a conserved linker region, and C and D constitute the association domain. αKAP is a muscle-specific protein, being abundantly expressed in skeletal muscle and at much lower levels in heart and uterus, but not in non-muscle tissues, such as liver [1, 67, 75].

Even though αKAP was found to have a sarcomeric distribution within skeletal muscle fibers, based on indirect immunofluorescence analysis [1], initial subcellular fractionation experiments did not support binding of αKAP to membranes [1]. However, Sugai et al. [75]

quickly demonstrated that αKAP was an integral membrane protein, and later Bayer et al. [2] demonstrated the localization of αKAP to SR in rat skeletal muscle, showing that αKAP was anchored to SR membranes via its N-terminal hydrophobic domain, and that it was responsible for targeting of CaMKII holoenzyme to SR (see also [69]). Finally, using highly purified SR membrane subfractions purified from rabbit skeletal muscles by isopycnic sucrose-density centrifugation, Damiani et al. [17, 19], and Colpo et al. [13] confirmed the subcellular localization of αKAP to SR.

The 11-amino acid insert III (KRKSSSSVQLM), introduced after a Lys (K) in the previous exon, leads to the formation of the sequence KKRK, which is a ca- nonical nuclear localization sequence (NLS), also found in SV40 T-antigen [35, 43, 69]. This opens the possibil- ity that αKAP may also be targeted to nuclei. However, it seems that the N-terminal hydrophobic sequence is dominant to the targeting information provided by the NLS [43, 69]. To the best of our knowledge, no evidence has been reported in support of the localization of αKAP in nuclei of native skeletal muscle fibers, either using immunofluorescence of cryostat sections or using fractions of purified nuclei. On the other hand, accumulation of αKAP within nuclei of COS-7 cells transiently expressing αKAP, occurred only when the hydrophobic amino-terminal segment had been deleted [78]. Using αKAP deletion mutants lacking the N- terminal hydrophobic domain, nuclear localization was found to be maintained in HeLa and HEK-293T cells, as well as in rat myotubes [58]. It has to be considered that none of the isoforms of CaMKII which contain the NLS and which are translocated into nuclei, i. e. α_B, and δ3

[43, 69], is expressed in skeletal muscle.

In agreement with results obtained at the mRNA level, a single protein isoform of αKAP of about 25-23 kDa was detected in skeletal muscle of adult mice [1] and of rat [2, 75]. In rabbit skeletal muscle, the situation seems to be more complicated. Two isoforms of αKAP of 23 and 21 kDa, respectively, are present in fast-twitch muscle SR in a 3:1 stoichiometry [19, 67]. The two isoforms, that are always detected together (see Figure 3) represent two splice variants of α CaMKII gene [19,

67]. Direct sequencing of the longer transcript demonstrated its identity with rat αKAP, while the shorter splice variant was found to lack the 33 bp-long insert, corresponding to the 11-amino acid sequence inserted in full-length αKAP. At variance with what found in fast- twitch muscle, in adult rabbit soleus only the 23 kDa isoform of αKAP could be detected [19, 67]. Thus far, there is no evidence that these differences in isoform composition of αKAP somehow affect the functional properties of CaMKII holoenzyme.

Finally, the expression of αKAP isoforms is develop- mentally regulated. The 21 kDa αKAP isoform is pecu- liar of immature muscle, and is the predominant αKAP variant expressed both in fast-twitch and slow-twitch muscle of 1-day-old rabbits, as well as in rat myotubes.

During postnatal maturation, a transition between the two isoforms occurs. The SR content of 23 kDa αKAP isoform progressively increases in both type of muscles, while the 21 kDa αKAP isoform progressively disap- pears from slow-twitch muscle SR. The isoforms transition was completed at three-weeks post-natal [67].

Distribution of full length subunits of CaMKII and of αKAP within the SR and membrane topology.

It is well known that SR is formed by two morpho- logically and biochemically specialized regions. The longitudinal SR, which is embedded with the Ca²⁺- pump and is functionally devoted to Ca²⁺ re-uptake from the cytosol during muscle relaxation, and junctional SR, which contains the RyR1, through which the intraluminal SR Ca²⁺ activating muscle contraction is released [see Figure 4].

Mostly thanks to subcellular fractionantion studies, it is now firmly established that the whole complement of full-length subunits is uniformly distributed within longitudinal and junctional SR, in rat skeletal muscle [2], as well as in fast-twitch ([19, 60], see also Figure 4) and slow-twitch [18, 65] muscle of the rabbit.

Concerning αKAP, using subcellular fractionation studies in rat skeletal muscle, Bayer et al. [2] reported that the distribution of αKAP within the different regions of SR matched that of CaMKII full-length subunits. Similar results were obtained for fast-twitch [17]

and slow-twitch [19] skeletal muscle of the rabbit. In Figure 4, the distribution of αKAP isoforms within various SR membrane subfractions was analyzed, vis a vis with that of undisputed protein markers of transverse tubules (α1 DHPR), longitudinal SR (SERCA1, 53 kDa glycoprotein) and junctional SR (calsequestrin, RyR1, triadin). It is apparent that distribution of αKAP isoforms does not parallel that of any marker, indicating a uniform distribution within SR. Taken together, these studies indicate that αKAP is ubiquitously distributed within SR of fast-twitch and slow-twitch muscles.

This conclusion was recently challenged by Nori et al.

[58], using indirect immunofluorescence as experimental approach and adult rat soleus muscle fibers made transgenic by electroporation with plasmid cDNA en-

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coding for αKAP tagged with haemoagglutinin (αKAP- HA1) as experimental model. In this study, αKAP-HA1 was found to be largely associated with free SR and only minor co-localization was observed with the junctional SR RyR1 protein marker. In this respect, it is important to say that, using CaM-affinity chromatography, αKAP (23 kDa isoform in slow-twitch, both 23-21 kDa isoforms in fast-twitch muscle) was always found to be stably integrated into purified CaMKII holoenzyme [19]. In addition the reported preferential localization of αKAP to longitudinal SR contrast with the ubiquitous distribution of CaMKII within longitudinal and junctional SR ([2, 17, 18, 19, 65]; see also Figure 3). Fur- thermore using highly-purified, vesiculated derivatives of junctional face membrane, i. e. vesicles deriving from the membrane domain of SR containing Ca²⁺-release channel/RyR1, Damiani et al. [19] clearly demonstrated the co-localization of αKAP with junctional SR protein markers, such as triadin. These observations suggest caution in the interpretation of data obtained only with morphological techniques.

Concerning the membrane topology of CaMKII, lim- ited proteolysis experiments carried out on right-side out SR vesicles demonstrated that CaMKII is localized on the cytoplasmic face of SR membrane, as expected [15, 16].

The amino acid sequence of αKAP suggests a two- domain structure of the protein: a short N-terminal hydrophobic domain and a large C-terminal domain identical to the association domain of CaMKII, and therefore expected to be cytoplasmic. In agreement with this model, αKAP associated to right-side out, purified SR membranes was found to be sensitive to mild tryp- tic digestion (E. Damiani, R. Sacchetto, unpublished observations).

Protein substrates of SR CaMKII

The identification of SR proteins phosphorylated by membrane-bound CaMKII was of crucial importance for understanding the regulative role of the kinase. Thus far, it is quite clear that in slow-twitch muscle, CaMKII is mainly involved to regulation of SR Ca²⁺-transport, whereas in fast-twitch muscle CaMKII seems to be more important for regulation of Ca²⁺-release from SR.

In addition, it might play a metabolic role, contributing to the regulation of glycogen synthase.

Slow-twitch muscle

It is known long since that, in heart and slow-twitch muscles, the activity of the SR Ca²⁺-ATPase (SERCA) is controlled by regulatory protein phospholamban (PLB) (see [52], for a recent review). PLB is phosphorylated by PKA at Ser-16 and by CaMKII at Thr-17.

Phosphorylation at Ser-16 seems to be mainly responsi- ble for in vivo β-adrenergic stimulation of the heart [52].

The most accepted model suggests that, in the non- phosphorylated form, PLB interacts with SERCA, re- ducing its affinity for Ca²⁺and thereby inhibiting the Ca²⁺-pump. When phosphorylated, interactions of PLB with SERCA are disrupted, and this leads to removal of inhibition and consequent activation of the Ca²⁺-pump.

It is important to point out that PLB is able to interact with all SERCA isoforms [48]. However, since the expression of PLB is restricted to slow-twitch muscles, this regulatory mechanism of SERCA activity is miss- ing in fast-twitch muscles.

The proposed model seems to invariantly work in cardiac muscle of all mammalian species so far investi- gated. On the other hand,there seems to exist a great heterogeneity in the level of expression of PLB between mammalian slow-twitch fibers. This was demonstrated in knock-out experiments showing that ablation of PLB was associated with a small, but significant increase in relaxation rates of mouse soleus [52]. In fact, PLB expression ranges from zero in the rat [18], to significant levels in the rabbit and even higher in humans [55]. Us- ing highly-purified SR subfractions from rabbit soleus.

Damiani et al. [18] carried out a thorough analysis of PLB phosphorylation state by phosphorylation site-

TT markers α1DHPR

LSR markers GP-53 170

53

triadin 95

jSR markers

γ CaMKII subunits

αKAPB

βM

αKAPA

72 60

23 21

B

R1 R2 R3 R4

200

116 97 77

55

RyR1

sarcalumeninHRC

SERCA1

CS

R1 R2 R3 R4

kDa

SR subfractions A

43

R1 R2 R3 R4

Stains All

Immunoblots

Figure 4. Analysis of distribution of CaMKII isoforms and of αKAP isoforms with SR, by subcellular fractionation studies. SR membrane subfractions were isolated from rabbit fast-twitch muscle, by isopycnic sucrose-density centrifugation, using the method of Saito et al. (1984), and are la- belled accordingly from top to bottom of the gradient. Proteins were resolved using linear gradient SGS-gels, and either stained with Stains all (panel A), or blotted onto nitrocellulose (panel B). Blots were incubated with specific an- tibodies. Based on the presence of α1 subunit of DHPR, fraction R1 is deemed to be most en- riched in transverse tubules. Fraction R2 con- tains SERCA1 and 53 kDa glycoprotein (GP- 53), in the absence of protein markers of TT and junctional SR, and istherefore judged to be largely derived from longitudinal SR. Fraction R4 is highest in content of calsequestrin (CS), RyR1 monomer and triadin, and, therefore, is mainly composed of terminal cisternae vesicles.

Viceversa, it is evident that β and γ isoforms of CaMKI, as well as both isoforms of αKAP, are uniformly distributed with SR subfractions.

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specific antibodies. The results obtained indicated that PLB, in isolated SR vesicles, was mainly phosphorylated at Thr-17, suggesting that in slow-twitch muscle, CaMKII might be more important than PKA in the regulation of Ca²⁺-transport.

For slow-twitch muscle, in addition to PLB CaMKII was shown to be able to phosphorylate the SERCA2a isoform of Ca²⁺-ATPase [18, 60, 90] at Ser-38 [82].

This explains its substrate specificity, since the fast- twitch SERCA1 isozyme lacks a phosphorylatable serine or threonine residue at position 38. Phosphorylation of SERCA2a resulted in a 2-fold increase in catalytic activity of the enzyme without alteration in its Ca²⁺- sensitivity. The increase in Ca²⁺-ATPase activity was matched by a 2-fold increase in Ca²⁺-transport activity of isolated SR vesicles [33, 34]. Thus, phosphorylation of SERCA2a by CaMKII resulted in an increase of Vmax. It should be pointed out that SERCA2a is not phosphorylated by PKA [33].

Fast-twitch muscle

The first indication that Ca²⁺-calmodulin-dependent phosphorylation of SR proteins might be involved in the regulation of Ca²⁺-release, came from two papers from Ikemoto’s laboratory. Using a heavy SR fraction isolated from rabbit fast-twitch muscles, Kim and Ikemoto [45]

showed that Ca²⁺-CaM-dependent phosphorylation of SR vesicles inhibited Ca²⁺-release from skeletal muscle SR.

These results were later duplicated, using SR membrane fractions from pig back muscle, a primarily fast-twitch muscle [46]. Subsequently, using single fibers from frog skeletal muscle, Wang and Best [87] reported inactivation of the SR Ca²⁺-release channel by an endogenous CaM- dependent protein kinase activity. These results were finally confirmed by Hain et al. [30], using TC vesicles from rabbit fast-twitch muscle fused to planar lipid bilayers to monitor single-channel currents.

These results prompted the hypothesis that the SR Ca²⁺-release channel of skeletal muscle might be phosphorylated by endogenous CaMKII. Early studies yielded contrasting results. In fact, Seiler et al. [71] reported that a rabbit skeletal muscle high Mr protein could be phosphorylated by an endogenous CaM kinase activity. Alternately, Kim et al. [46] did not observe any phosphoprotein in the 200-450kDa range of molecular weight. However, Witcher et al. [88] later clarified that the high Mr phosphorylated protein described in Seiler’s paper did not correspond to RyR1 (skeletal muscle) isoform, but was a protein unrelated to RyR1, migrating in SDS-PAGE at the bottom edge of RyR1 (see also ref.

[74]). The presence in SR membranes of a protein component having electrophoretic mobility similar to that of RyR1 and phosphorylated even in the absence of a RyR1 immunoreactive component (see the Discussion section of ref. [74]) generated much confusion, and seems to explain some discrepancies in the interpretation of experimental results obtained in different labora- tories [12], concerning this important issue.

Since then, several papers have been published, showing that the RyR1 was not significantly phosphorylated by endogenous CaMKII in isolated SR from fast and slow-twitch muscles of the dog [77], in porcine fast- twitch muscle [74], as well as in rabbit skeletal muscle [13, 15, 16, 49]. These results are even more striking, in view of the fact that the cardiac isoform of RyR [RyR2]

is know to be a remarkable substrate of endogenous SR- bound CaMKII [38, 71, 74, 77, 88].

Phosphorylation of RyR1 by endogenous protein kinase(s) activity was reported by Varsanyi and Meyer [84]. However, it ought to be said that, in this work, phosphorylation experiments were carried out [see ref.

36 for detailed experimental conditions] in the presence of 1 mM EGTA and without exogenously added CaM, i.

e. under experimental conditions very unsuitable for activation of SR-bound CaMKII.

Suko et al. [76] and Hain et al. [30] reported phosphorylation of RyR1 by exogenously added CaMKII.

In the first paper, exogenous CaMKII was purified from heavy SR, whereas in the latter paper the origin of purified CaMKII was not specified, even though it was probably of brain origin. If so, CaMKII had a subunit composition greatly different from that of skeletal muscle, SR-bound CaMKII. This might explain why phosphorylation of TC vesicles by exogenous CaMKII activated the channel, whereas phosphorylation by endogenous CaMKII inhibited RyR1 channel activity.

The artificial nature of this approach makes interpretation difficult.

While phosphorylation of RyR1 by endogenous CaMKII seems to be negated by the weight of experimental work, others substrates have been identified in junctional SR. Among them, histidine-rich Ca²⁺-binding protein [14, 39] (HCR, see ref. [40]) and of triadin.

Phosphorylation of HRC is a well established fact [13, 15, 16]. Given the cytoplasmic location of SR-bound CaMKII [15], this observation strongly argues for a cytoplasmic location of HRC.

Phosphorylation of triadin is most interesting, since it is well known that triadin interacts structurally and functionally with RyR1 [44, 50]. What makes this observation even more interesting, is the fact that phosphorylation site was identified [13] in the triadin cytoplasmic domain (amino acid residues 1-47), i. e. the triadin domain that interacts with RyR1 in a Ca²⁺- dependent fashion, decreasing the open probability of the Ca²⁺-channel [28]. This is exactly what one would expect, given the cytoplasmic localization of CaMKII on SR membrane [15]. The hypothesis, therefore, is that the interaction of triadin with RyR1 may be regulated by Ca²⁺-CaM-dependent phosphorylation of triadin, in the same way that interaction of PLB with SERCA is regulated by phosphorylation of PLB. It should be noted that, thus far, no other protein kinase has been reported to phosphorylate triadin.

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As mentioned before, previous studies showed that endogenous, CaMKII-dependent phosphorylation inhibited RyR1 calcium channel activity. Recently, this tradi- tional view was challenged by two reports. Using isolated SR membranes from rabbit skeletal muscle incor- porated into lipid bilayers, Dulhunty et al. [22] reported irreversible activation by ATP of skeletal RyR1 calcium channel activity, which was prevented by a CaMKII inhibitory peptide or by CaMKII inhibitor KN-93. These results suggested that RyR1 activity was positively modulated by endogenous CaMKII-dependent phosphorylation of SR proteins. On the other hand using single isolated fast-twitch fibers from mouse flexor digito- rum brevis muscle, Tavi et al. [80] reported that injec- tion of CaMKII inhibitory peptide decreased tetanic [Ca²⁺]i. From their experiment, the authors concluded CaMKII-induced phosphorylation facilitates SR Ca²⁺- release in the basal state and during repeated contrac- tions. Clearly, more experimental work is required to clarify this important issue.

Besides high Mr phosphoprotein, triadin and histidine- rich Ca²⁺-binding protein, other protein substrates have been described of 88 kDa [12, 15], and in the molecular weight range of 37-30 kDa [10, 12, 15, 60]. While the identification of the latter peptides remains still obscure, it has recently been assessed that the 88 kDa phosphorylatable protein is glycogen synthase (GS) [19]. The association of GS to SR in form of a protein-glycogen complex is known long since [9]. However, we have recently found [20], that association of GS to longitudinal SR fragments prepared from rabbit fast-twitch muscles, is strongly dependent on the isolation procedure.

When SR membranes were prepared from untreated rabbits, GS was recovered in the cytosolic fraction.

Conversely when SR subfractions were isolated from rabbits injected with β−propranolol (a β-adrenergic receptor blocker), GS was found to remain associated with SR, in form of a complex with glycogen and protein phosphatase 1 catalytic subunit. Furthermore, the GS associated with SR was found to be highly phosphorylated by endogenous SR-bound CaMKII. In view on the regulatory role that protein phospho- dephosphorylation exerts on regulation of GS activity [63], this preliminary observation is of great potential interest. Is is noteworthy that, in rabbit liver, CaMKII- dependent phosphorylation of GS results in a partial inactivation of the enzyme [59]. Furthermore, the observation of a role of CaMKII in regulation of glycogen metabolism is in full agreement with a recent report, indicating a role for CaMKII in insulin stimulated glucose transport in skeletal muscle [89]. It is an appealing hypothesis that CaMKII might participate in regulating glucose metabolism, in particular during to physical exercise.

Finally, it was recently demonstrated that, in rabbit skeletal muscle SR, the 21 kDa αKAP isoform acts as a substrate of SR-bound CaMKII [17, 19]. The fact that

the 21 kDa isoform, but not the 23 kDa isoform, of αKAP can be phosphorylated, is likely due to the absence of the 11-amino acid sequence in 21 kDa αKAP splice variant. This, in turn, creates a novel CaMKII phosphorylation site [19]. Since 21 kDa αKAP isoform is expressed only in rabbit fast-twitch muscle [19, 67], phosphorylated αKAP is lacking in the isolated SR from rabbit soleus muscle. The functional relevance of this phosphorylation to the function of the holoenzyme, or in targeting of CaMKII to SR remains to be established.

Conclusions

In the past twenty years, biochemical-functional characterization studies of SR-bound CaMKII system provided experimental evidences that this protein kinase plays an important role in the regulation of Ca²⁺- homeostasis in skeletal muscle fibers. In slow-twitch muscle, SR-bound CaMKII mainly regulates SR Ca²⁺- uptake, through phosphorylation of PLB and of SERCA2a. In fast-twitch muscle, CaMKII controls SR Ca²⁺-release channel activity, even though controversy is still raging, concerning the mechanism(s) and the positive or negative effects. In addition, evidence is ac- cumulating, suggesting a role of SR-bound CaMKII in regulation of glucose metabolism. It is tempting to speculate that activation of CaMKII during muscle con- tractions, might provide an insulin-independent mechanism for regulating glucose metabolism. Further studies are needed to substantiate the functional significance of SR-bound CaMKII, in relation to this problem.

Acknowledgements

We thank Prof. Arianna Donella-Deana and Prof.

Roger A. Sabbadini for critical reading of the manu- script.

Address correspondence to:

E. Damiani, Dipartimento di Scienze biomediche sperimentali, Università di Padova, viale G. Colombo 3, 35121 Padova, tel. 0498276038, Email damiani@civ.

bio.unipd.it.

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