Calmodulin-Dependent Protein Kinase II

C

H

A

P

T

E

R

11 Calcium/Calmodulin- Dependent Protein Kinase II HAROLD A. SINGER, S. THOMAS ABRAHAM, and CHARLES M. SCHWORER Sigfried and Janet Weis Center for Research Geisinger Clinic Danville, Pennsylvania

II. STRUCTURAL PROPERTIES OF CALMODULIN-KINASE II

I. INTRODUCTION Ca 2+/calmOdulin (Ca 2+/CaM)-dependent protein kinases are important mediators of signal transduction events triggered by stimuli that increase intracellular levels of free [Ca2+]. Phosphorylase kinase and myosin light chain kinase (MLCK) are two examples of Ca2+/CaM-dependent kinases that have specific substrates and well-defined functions. Several Ca2+/ CaM-dependent kinases have been identified that phosphorylate multiple substrates in vitro and are likely to have diverse cellular functions analogous to other multifunctional kinases, such as the cyclic nucleotidedependent protein kinases and protein kinase C (PKC). Ca 2+/calmodulin-dependent protein kinase II, referred to here as CaM-kinase II, is the best known of these multifunctional CaM-kinases. Much of the current state of knowledge relating to this protein kinase has resulted from the availability of readily purified kinase from brain, where it accounts for as much as 2% of the total protein in certain regions (Erondu and Kennedy, 1985). Interest in brain CaM-kinase II has been fueled by its implicated involvement in such critical functions as synaptic transmission and memory (Hanson and Schulman, 1992). However, CaM-kinase II is ubiquitous, and as insight into its unique structural and autoregulatory features has grown, investigators have become interested in its functional roles in other tissues. In this chapter we will review these structural and regulatory properties with emphasis on CaM-kinase II and its cellular activation in smooth muscle.

BIOCHEMISTRYOF SMOOTHMUSCLECONTRACTION

A. Holoenzyme and Subunit Structure CaM-kinase II has been purified from mammalian brain and a number of peripheral tissues such as liver and heart (see Colbran and Soderling, 1990, for review). In each case it has been described as a large multimeric protein of approximately 300-600 kDa composed of 50- to 60-kDa kinase subunits. Four primary subunits (~, [3, 8, ~/) were first identified and cloned from rat brain cDNA libraries (Lin et al., 1987; Bennett and Kennedy, 1987; Tobimatsu et al., 1988; Tobimatsu and Fugisawa, 1989). The domain structures of the known subunits and their calculated molecular masses are shown in Fig. 1A. Multiple alternatively spliced CaM-kinase II subunits that are closely related to the rat oL-subunit (77% amino acid identity) have also been identified in Drosophila (Cho et al., 1991; Ohsako et al., 1993), indicating the kinase is strongly conserved across species. The first 315 amino acids (AA) are highly conserved among the subunits and comprise the "catalytic/regulatory" domain containing a consensus ATP binding sequence, catalytic, and regulatory regions. A variable region separates the catalytic/regulatory domain from a C-terminal "association" domain, which is approximately 150 AA in length and also highly conserved among subunits. The 8-subunit originally cloned from brain is distinguished structurally from the other subunits by a unique 21-AA carboxyl terminus (Tobimatsu and Fu-

143

Copyright 9 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

| 44

HAROLD A. SINGER, S. THOMAS ABRAHAM, AND CHARLES M. SCHWORER

A

Subunit ATP

O

I ~

Cat.

Reg.

W

~1

I

(A.A.)

(kDa)

478

54.1

527

59.0

/ m

h9

@

I

Catalytic/Regulatory Domain B

ATP

Cat.

Association Domain

Reg.

association

Pt 291 301 PI PI 311 319 MHRQETVDCLKKFNARRKLKGAILTTMLATRNFSGGKSG

281

Autophos.

Autoinhib. CaM-Binding

FIGURE 1 (A)CaM-kinaseIIsubunitstructures. Open boxes indicate conserved domains and solid boxes variable domains. Shaded areas designate conserved ATP binding (ATP), catalytic (Cat.), and regulatory (Reg.) domains. The conserved association domain is required for the formation of the multimeric holoenzyme. Sizes of the full-length subunits are indicated by the number of amino acids in the sequence (A.A.) and calculated molecular mass (kDa). (B) CaMkinase II regulatory domain. Overlapping autoinhibitory (Autoinhib.) and calmodulin (CAM) binding domains and known autophosphorylation sites are indicated. Thr 286 has been identified as the primary autophosphorylation site (Autophos.) conferring Ca 2+/CaM-independent activity to the kinase. Secondary autophosphorylation events include Thr 3~ which in vitro results in a decreased binding affinity for CaM.

jisawa, 1989). Proteolytic cleavage of subunits in the regulatory region (Colbran et al., 1988; Yamagata et at., 1991) or expression of mutant kinase subunits truncated in the regulatory region (Cruzalegui et al., 1992) generates a constitutively active monomeric kinase activity, hence the name for the conserved "association" domain, which is inferred to be required for formation of the holoenzyme. Electron microscopic imaging of purified brain CaM-kinase II molecules suggests that the holoenzyme has the shape of a rosette (similar to that depicted in Fig. 4), consistent with the association of 8-10 "dumbbell"-shaped kinase subunits (Kanaseki et al., 1991). An additional level of structural complexity within

the CaM-kinase II subunits has become apparent with the identification of a number of subunit variants. Alternatively spliced forms of the ~-subunit (~, ~') (Bulleit et al., 1988), ~-subunit (~, ~-33) (Benson et al., 1991), 8-subunit (~1-4) (Schworer et al., 1993), and ~-subunit (~A-C) (Nghiem et al., 1993; Zhou and Ikebe, 1994), differing in variable domain nucleic acid sequence, have been identified. In addition, Mayer et al. (1993) have described 8-subunit variants lacking the 8-specific C-terminal sequence. Figure 2 depicts the domain structure of 8-isoforms deduced from rat nucleic acid sequences identified by reverse transcriptasepolymerase chain reaction (RT-PCR) cloning of mRNA from various rat tissues (Schworer et al., 1993;

11. Calcium/Calmodulin-Dependent Protein Kinase II

INNKANVVTSPKENIPTPALEPQTTVIHNPDGNK Domains."

A TP

CA T

Reg. ', ',', ', Association

81

I83 i i i i

K~kssssv6~M

ID

I-

FIGURE 2 Structure of 8-subunit variants deduced from PCRamplified target sequences of 8-subunit cDNA from rat brain, VSM, and skeletal muscle (Schworer et al., 1993). The deduced amino acid sequence in the variable domain is indicated. The variable domain is deleted in 82, substituted in 83, and truncated in 8 4 by deletion of the 81 variable domain sequence EPQ...GNK. 85_8 signifies 8-subunit variants equivalent to 81_4 but lacking the unique C terminus (Mayer et al., 1993).

Mayer et al., 1993). There are interesting similarities between these and the other reported subunit splice variants, indicating some conservation of structure in the variable region. For example, oL-subunits and the 82-subunit completely lack variable region sequence, and a 15-AA sequence (-E-P-...-N-K-) at the end of the variable domain in 81 is conserved in the f~- and ~/-subunits, but missing in ~, ~', and ~S2_ 4. The length of the subunit variable region may determine or influence the number of subunits comprising the holoenzyme. CaM-kinase II purified from rat forebrain has an ~/f~ subunit stoichiometry of 3:1 and occurs primarily as homomultimers of 10 c~-subunits, whereas CaM-kinase II from cerebellum, which is enriched in the larger f3-subunit (oL/~3 = 1:4), occurs as homomultimers averaging 8 13-subunits (Kanaseki et al., 1991). In other brain regions and in peripheral tissues where the expression of multiple subunits is more balanced, it is likely that heteromultimers also form (Yamauchi et al., 1989). The number and composition of subunits could affect certain kinetic properties of a CaM-kinase II holoenzyme, such as the cooperative intersubunit autophosphorylation described in Section III.B. In many tissues such as brain, heart, and liver, CaM-kinase II is mainly cytosolic, whereas in others a variable amount has been reported to be associated with membranes or particulate fractions (Colbran and Soderling, 1990). CaM-kinase II has been localized in junctional sarcoplasmic reticulum from skeletal

145

muscle (Chu et al., 1990), brush border membranes from ileum (Cohen et al., 1990), and cytoskeletons from postsynaptic densities (Sahyoun et al., 1985), ileal enterocytes (Matovcik et al., 1993), and adrenal cortical cells (Papadopoulus et al., 1990). The structural features in the holoenzyme or the individual subunits that are responsible for directing the localization are only beginning to be considered experimentally. Interestingly, three CaM-kinase II subunits (oL-33, 83, and ~/A) share a conserved 11-AA variable domain insert (K-R-K-S-S-S-S-V-Q/H-L/M-M), which recently has been shown to be involved in nuclear targeting (Srinivasan et al., 1994). Using cotransfection of CaM-kinase II subunit mutants containing or lacking this sequence, it was demonstrated that the amount of CaM-kinase II localizing in the nucleus of overexpressing COS cells and cardiomyocytes was dependent on the relative proportion of the transfected subunits containing this sequence. Though it remains to be shown that this sequence is involved in controlling nuclear localization of CaM-kinase II in normal cells containing physiological concentrations of CaM-kinase II, this study provides direct evidence that the CaM-kinase II subunit variable domain and holoenzyme subunit composition are important factors in targeting the kinase to specific subcellular localizations and substrates. B. Tissue Distribution of Calmodulin-Kinase II Subunits

The subunit structure of native CaM-kinase II isozymes and the expression of subunits at the level of mRNA have not been studied in detail in tissues other than brain, where the c~- and ~-subunit mRNA is enriched compared to 8- and ~/-subunit mRNA (Tobimatsu and Fujisawa, 1989). In brain, ~-, 13-,and 13'-subunits are differentially expressed both regionally and developmentally (Kelly et al., 1987; Tobimatsu and Fujisawa, 1989). Based on Northern analysis, skeletal muscle has been reported to express primarily f~-, 8-, and ~/subunit mRNA, whereas cardiac muscle, smooth muscles, and other peripheral organs appear to express 8- and ~/-subunits (Tobimatsu and Fujisawa, 1989). RT-PCR analysis of mRNA from various tissue sources indicates that the full-sized 8-subunit is expressed only in brain (Schworer et al., 1993). Therefore, the reported distribution of the 8-subunit mRNA in peripheral tissues reflects the differential distribution of the smaller 82, 8 3 and 8 4 variants. For example, the RT-PCR analysis indicates that aortic smooth muscle expresses 8 2 and 83, cardiomyocytes primarily 83, and skeletal muscle the 84-isoforms (Schworer et al., 1993). Confirmation of the subunit composition in a given

146

HAROLD A. SINGER, S. THOMAS ABRAHAM, AND CHARLES M. SCHWORER

CaM-kinase II holoenzyme requires that the subunits and alternatively spliced variants be identified using specific antibodies. Given the high degree of homology between the subunits and variants, production of such reagents can be expected to be difficult. To date, ~-subunit-specific antibodies have not been described. However, monoclonal antibodies specific for c~- and f~-subunits are available and have been used to identify these subunits in brain holoenzymes (Baitinger et al., 1990). These antibodies fail to cross-react with CaM-kinase II from rat aortic vascular smooth muscle or swine cartoid artery, consistent with a lack of oL-and ~-subunit mRNA expression in these tissues (Singer, H. A., unpublished observations). Expression of varying sized 8-subunit variants has been documented at the protein level in rat brain and aortic medial smooth muscle extracts by immunoblotting with an antipeptide antibody (CK2-Delta) that specifically recognizes 8-subunit variants containing the unique 21-AA C terminus (Schworer et al., 1993). A 53-kDa 8-subunit (82) and approximately 80% of the total CaM-kinase II activity can be immunoprecipitated from lysates of cultured rat aortic vascular smooth muscle (VSM) cells with the CK2-Delta antiserum (Fig. 3). In contrast, only a few percent of the total CaM-kinase II activity and multiple subunits (50, 53, 58 kDa) are immunoprecipitated from rat brain lysates with CK2-Delta. FiftykDa oL-subunits and 60-kDa 13-subunits are detected by 125I-labeled calmodulin overlay in preparations of purified rat brain CaM-kinase II (first lane) and in immune complexes from rat brain lysates formed with a subunit nonspecific antipeptide antibody (CK2-CAT)

FIGURE 3 Immunoprecipitation of CaM-kinase II from rat brain and cultured rat aortic VSM cell (RAVSM) lysates with antibodies specific to the 8-subunit C terminus (CK2-Delta) or a conserved sequence in the catalytic/regulatory domain (CK2-CAT). The immune complexes were subjected to SDS-PAGE and transferred to immobilon membranes. CaM-kinase II subunits were detected by an overlay with 125I-labeled calmodulin and autoradiography. The left lane is a control containing partially purified rat brain CaMkinase II (CK2), which was not immunoprecipitated.

directed to a conserved sequence in the catalytic domain (last lane). The low efficiency of immunoprecipitation from brain compared to VSM cell extracts with CK2-Delta is consistent with the bulk of the brain CaM-kinase II being composed of e~- and f~-subunits. The appearance of multiple subunits in the immune complexes from rat brain indicates either the expression of multiple 8-subunits (i.e., 81 and 82) and/or immunoprecipitation of hetermultimers containing both 8- and non-8-subunits.

C. Related Calmodulin-Kinases (CalmodulinKinases I, III, IV, V) Several other Ca 2+/CaM-dependent serine/ threonine kinases have been identified that can be distinguished from CaM-kinase II on the basis of native size, substrate specificity, and autophosphorylation properties. One of these is CaM-kinase Gr or IV, which is a multifunctional kinase found primarily in brain (Means et al., 1991). CaM-kinase IV has a peptide substrate specificity that is similar to that of CaM-kinase II, and it also undergoes Ca2+/CaM-dependent autophosphorylation on a serine residue (Frangakis et al., 1991). However, autophosphorylation of CaM-kinase IV expressed in insect cells does not result in the generation of Ca2+/CaM-independent activity (Cruzalegui and Means, 1993). CaM-kinase IV is only 30-40% identical to a CaM-kinase II subunit and is monomeric with an estimated molecular mass of 61-67 kDa by SDSPAGE depending on its source (Means et al., 1991; Cruzalegui and Means, 1993). Two other classes of CaM-dependent kinases, CaM-kinases Ia and Ib (DeRemer et al., 1992; Picciotto et al., 1993) and CaMkinase III (Nairn et al., 1985), can readily be distinguished from CaM-kinase II by their native size and restricted substrate specificity. Although these protein kinases also autophosphorylate, this is not associated with a transition to a Ca2+/CaM-independent activity. A monomeric (37-41 kDa) Ca2+/calmodulin dependent kinase activity designated CaM-kinase V, which is similar catalytically to CaM-kinase II, has been isolated from rat cerebrum (Mochizuki et al., 1993). CaM-kinase V undergoes Ca2+/CaM-dependent autophosphorylation, but this has not yet been shown to lead to formation of autonomous activity.

III. Regulation of Calmodulin-Kinase II Activity A. A c t i v a t i o n by Ca 2+ ] Calmodulin

The Km of purified CaM-kinase II for calmodulin has been reported to be in the range of 20-100 nM at saturating [Ca 2+ ] with one calmodulin bound per sub-

11. Calcium/Calmodulin-Dependent Protein Kinase II

unit (Colbran and Soderling, 1990; Schulman and Hanson, 1993). This calmodulin affinity is about a factor of 10 lower than that for other calmodulin-activated kinases like MLCK (Kamm and Stull, 1989). Analysis of (x- and 13-subunits expressed in mammalian cells indicates that calmodulin affinity varies somewhat between these subunits (Yamauchi et al., 1989). However, no significant differences were found in another study comparing expressed (x- and 8-subunits (Edman and Schulman, 1994). Based on homology to the calmodulin binding domains in phosphorylase kinase and MLCK, a putative regulatory region in CaMkinase II was identified (Fig. 1B) and peptides based on AA sequence in this region were designed and assessed for their ability to inhibit calmodulin binding and activation of CaM-kinase II (Payne et al., 1988). The calmodulin binding domain has been localized to amino acids 296-309 and a peptide corresponding to this sequence has been shown to inhibit Ca2+/CaM dependent activation of CaM-kinase II with an IC50 of 57 nM and CaM-activated phosphodiesterase with an IC50 of 3.4 nM. Similar studies evaluating the ability of peptides to competitively inhibit the activity of autophosphorylated Ca 2+/CaM-independent CaM-kinase II identified an autoinhibitory domain corresponding to amino acids 291-302 (Payne et al., 1988; Colbran and Soderling, 1990). Additional inhibitory potency was gained by extending the peptides to amino acid 281, though inhibition became noncompetitive and involved inhibition of ATP binding (Colbran and Soderling, 1990). Other studies using truncated mutants, site-directed mutagenesis, and molecular modeling approaches (Cruzalegui et al., 1992) have generally confirmed the C-terminal boundary of the calmodulin binding domain and position of the autoinhibitory domain as indicated in Fig. lB.

147

dependent protein kinases and could be of physiological importance, for example, by effecting prolonged intracellular responses to transient Ca 2+ signals. Therefore, the mechanisms, sites, and functional consequences of CaM-kinase II autophosphorylation events have been intensively investigated using peptide mapping and sequencing approaches and more recently site-directed mutagenesis. It is now known that generation of activatorindependent CaM-kinase II activity requires autophosphorylation of a specific threonine residue (Thr 286) in the autoinhibitory domain (Schworer et al., 1988; Thiel et al., 1988; Hanson et al., 1989). Autophosphorylation on this residue is via a highly cooperative intraholoenzyme intersubunit reaction that occurs only between activated (Caa+/CaM bound) subunits (Hanson et al., 1994; Mukherji and Soderling, 1994). In addition to generating autonomous activity, autophosphorylation of CaM-kinase II on Thr 286 has been shown to result in calmodulin "trapping" as a consequence of nearly a thousandfold decrease in calmodulin off-rate (Meyer et al., 1992). This activation scheme is summarized in Fig. 4. Autophosphorylation-dependent calmodulin trapping and enzymatic activity in the absence of Ca2+/CaM provide mechanisms for prolonging kinase activity beyond the dura-

~

[Ca2*]i ~ CaM

1. inactive

2. active

.~-ATP

B. Autophosphorylation of Calmodulin-Kinase H

Early studies of CaM-kinase II indicated that the kinase subunits autophosphorylated with stoichiometries estimated to be as high as 4 mol Pi/mol subunit (Ahmad et al., 1982). Ca2+/CaM-dependent autophosphorylation was subsequently shown to result in the generation of activator-independent kinase activity to a maximum level of 40-70% of total Ca2+! CaM-stimulated activity (Miller and Kennedy, 1986; Schworer et al., 1986; Lou et al., 1986; Lai et al., 1986). Once CaM-kinase II is activator independent or "autonomous," additional autophosphorylation can take place in the absence of Ca2+/CaM (Miller and Kennedy, 1986; Hashimoto et al., 1987). These properties distinguish CaM-kinase II from other Ca2+/CaM -

autophosphorylation

ph~sp~atase I

~ ADP

ef

00 4. partially active

3. active

FIGURE 4 Model for activation and autophosphorylation of a CaM-kinase II holoenzyme based on Hanson et al. (1994). 1-2: Multimers of CaM-kinase II subunits are activated by increasing concentrations of free intracellular Ca 2+ [(Ca2+]i) and association of Ca 2+/CaM complexes. 2-3: Activated subunits (solid) with bound Ca 2+/CAM are autophosphorylated on Thr 286 (P) in a cooperative intraholoenzyme intersubunit reaction. 3-4: Phosphorylated subunits have an increased affinity for CaM and are partially active (shaded circles) in the absence of bound Ca 2+/CAM. 4-1: Dissociation of Ca 2+/CaM and the action of phosphoprotein phosphatases return autophosphorylated subunits to the inactive state.

148

HAROLD A. SINGER, S. THOMAS ABRAHAM, AND CHARLES M. SCHWORER

tion of the Ca 2+ signal and for responding in a frequency-dependent manner to pulsatile changes in free intracellular Ca 2+ (Hanson et al., 1994). Once subunits are autophosphorylated on Thr 286 in vitro, Ca 2+ chelation and dissociation of CaM result in the autophosphorylation of additional serine and threonine residues, including Thr 3~ and/or Thr 3~ in the calmodulin binding domain (Miller and Kennedy, 1986; Hashimoto et al., 1987; Patton et al., 1990). Phosphorylation of Thr 3~176 blocks rebinding of CaM and this is reflected by a loss of total Ca 2+/CaM-dependent activity to a level equal to the activator-independent activity. Autophosphorylation of Thr 3~ in this manner also appears to be an intraholoenzyme intersubunit reaction (Mukherji and Soderling, 1994). Under certain experimental conditions it has been possible to demonstrate the slow autophosphorylation of Thr 3~ and concomitant loss of CaM-kinase II activity without addition of activators and prior autophosphorylation of Thr 286 (Colbran, 1993). In this case, Thr 3~ autophosphorylation appears to be catalyzed by an intrasubunit reaction (Mukherji and Soderling, 1994). C. Substrate Specificity

Analysis of the sequences around phosphorylated residues and analysis of the phosphorylation kinetics using a series of small peptide substrates (Pearson et al., 1985) indicate that a minimal consensus phosphorylation sequence for CaM-kinase II is -R-X-X-S/T-. However, there are exceptions to this, including the sequence around the Thr 3~ autophosphorylation site. As would be expected given this relatively loose consensus phosphorylation sequence, numerous proteins have been shown to be substrates for CaMkinase II in vitro (Colbran and Soderling, 1990; Schulman and Hanson, 1993), including smooth muscle regulatory proteins such as LC20, caldesmon, calponin, and MLCK (see Section VI). Few of these potential substrates have been shown to be phosphorylated specifically by CaM-kinase II in vivo. The difficulties in determining in vivo substrates for CaM-kinase II are not unique for this protein kinase and include a lack of specific tools and approaches for manipulating kinase activity in situ, the requirement for assessing sitespecific phosphorylation of the target proteins, and the presence of other multifunctional kinases that are Ca 2+ dependent and share substrate specificity, such as CaM-kinase IV and PKC. Tools that have been used to identify physiological substrates and functions for CaM-kinase II include inhibitory peptides based on AA sequences in the regulatory domain (Payne et al., 1988) and specific chemical inhibitors such as KN-62 and KN-93. KN-62 inhibits CaM-kinase II with a Ki of

0.9 ~M with no significant inhibitory effects on MLCK, cAMP-dependent protein kinase, or PKC (Tokumitsu et al., 1990). The mechanism of inhibition by KN-62, and a more water-soluble derivative KN-93 (Sumi et al., 1991), appears to be by competitive inhibition of calmodulin binding. Consequently, the autophosphorylated form of the kinase is not affected by the inhibitor (Tokumitsu et al., 1990). KN-62 has been used in intact PC-12 cells to inhibit CaM-kinase II autophosphorylation and generation of autonomous activity following stimulation by ionophores or KC1 depolarization (Tokumitsu et al., 1990).

IV. ACTIVATION A N D AUTOPHOSPHORYLATION OF CALMODULINKINASE II in Situ There is substantial evidence that CaM-kinase II autophosphorylation events, which have been characterized in vitro, also occur in situ following activation of intact cells with stimuli that increase intracellular free Ca 2+. Increases in activator-independent CaM-kinase II activity, reflecting activation of CaM-kinase II and autophosphorylation of Thr 286, have been demonstrated in cerebellar granule cells (Fukunaga et al., 1989), GH 3 cells (Jefferson et al., 1991), and PC-12 cells (MacNicol et al., 1990) stimulated by agonists or ionophores. CaMkinase II 8-subunits undergo phosphorylation in intact cultured VSM cells stimulated with the Ca 2+ ionophore ionomycin, an effect that can be potentiated by preincubating the cells with the protein phosphatase inhibitor okadaic acid (Fig. 5A). Increases in Ca2+/ CaM-independent CaM-kinase II activity, reflecting activation and autophosphorylation of CaM-kinase II in situ, can also be demonstrated in lysates from ionomycin-stimulated VSM cells in response to increases in free intracellular calcium ([Ca 2+ ]i), which are well within a physiological range (i.e., 150-400 nM) (Fig. 5B). Physiological stimuli such as angiotensin II, vasopressin, ATP, and platelet-derived growth factor (PDGF) also stimulate variable increases in autonomous CaM-kinase II activity in parallel with their abilities to increase [Ca2+]i over this range (Abraham, S. T., unpublished data). In these experiments, CaM-kinase II activity was assayed using a synthetic peptide substrate (autocamtide-2; KKALRRQETVDAL) that corresponds in part to the conserved autophosphorylation site in the known CaM-kinase II subunits. Autocamtide-2 has proven to be a useful substrate for measuring CaM-kinase II activity since it is not a substrate for cAMP-dependent protein kinase, smooth muscle MLCK, or PKC under the activation conditions used (Hanson et al., 1989; Schworer, C. M., unpublished data).

11. Calcium/Calmodulin-Dependent Protein Kinase II

| 49

FIGURE 5 (A) Autophosphorylation of CaM-kinase II 8-subunits in cultured rat aortic VSM cells. CaMkinase II was immunoprecipitated with a 8-subunit-specificantibody (CK2-Delta)from lysates of 32p-labeled cells following stimulation of the intact cells for the indicated times with ionomycin(1 ~M). Pretreatment of the cells with the protein phosphatase inhibitor okadaic acid (O.A.; 1 ~M) potentiated the phosphorylation response. (B) Correlation between [Ca2+]i (measured by fura-2 fluorescence) and autophosphorylationdependent generation of Ca2+/calmodulin-independent kinase activityin suspensions of cultured rat aortic VSM cells stimulated by varying concentrations of ionomycin.

V. C A L M O D U L I N - K I N A S E II ISOZYMES IN S M O O T H MUSCLE A. Caldesmon Kinase

Previous reports describing CaM-kinase II in smooth muscle have related primarily to a Ca 2+/CAMd e p e n d e n t kinase activity that copurifies with smooth muscle caldesmon (Ngai and Walsh, 1985; Scott-Woo and Walsh, 1988; Abougou et al., 1989). Evidence that the caldesmon kinase activity was related to CaMkinase II included close similarities in activator dependencies and substrate specificity using caldesmon (Abougou et al., 1989) and synapsin as substrates (Scott-Woo et al., 1990). Protein bands in the 50-kDa range, consistent with k n o w n CaM-kinase II subunit sizes, were identified on Western blots of purified cald e s m o n with an antipeptide antibody to a conserved sequence in CaM-kinase II subunits (Abougou et al., 1989). Dissociation of the endogenous Ca2+/CaM d e p e n d e n t kinase activity and caldesmon was ultimately achieved by treatment of a myofibrillar fraction (from avian gizzard) with 30 mM Mg 2+ and resolution by anion-exchange chromatography (Ikebe et al., 1990). The purified kinase was concluded to be a smooth muscle isozyme of CaM-kinase II based on its catalytic properties (using caldesmon as the primary

substrate), its ability to autophosphorylate resulting in generation of Ca 2+/CaM-independent kinase activity, and some physical properties, such as the apparent subunit molecular weight on SDS-PAGE gels (Ikebe et al., 1990). However, the native size of this kinase, the structural basis for its apparent tight binding to caldesmon, and its relationship to CaM-kinase II holoenzymes in other smooth muscle cellular compartments have not been determined. B. Arterial S m o o t h Muscle Calmodulin-Kinase H Ca 2+/CaM-dependent kinase activity is also readily measured in low-ionic-strength extracts from rat aortic (Fig. 5B) (Schworer et al., 1993) and swine carotid arterial smooth muscle (Fig. 6) using the peptide substrate autocamtide-2, consistent with a cytosolic distribution. The specific activity of the soluble kinase is approximately one-tenth of that in an analogous fraction of rat or swine forebrain (Schworer et al., 1993), where the average concentration of CaM-kinase II subunits has been estimated to be 1% of total brain protein. On the basis of a native size of 550-650 kDa estimated by gel filtration chromatography, peptide substrate specificity, autophosphorylation properties, and crossreactivity with subunit nonselective anti-CaM-kinase

HAROLD A. SINGER, S. THOMAS ABRAHAM, AND CHARLES M. SCHWORER

150

m.

320 1 - e - +CaM 280

A

s

5000

:)t

A c

O. o~ 4) .._ Ca (g O

=

240

-a-

+EGTA

9o

A(280)

200

4000

A

"7 :E

NaCI

O 160

Z ,....,

"~,

120

A o

<(

80

3000

2000

E

ol .'" "

1000

W//M A

A-2

B +

~6ra.

<

B

0

o

B-

C

A's+B's

0

10

20

30

40

50

60

Extract Fraction N u m b e r FIGURE 6 (A) Sequential extraction of Ca2+/CaM-dependent autocamtide-2 kinase activity from swine carotid artery medial smooth muscle. Twenty-five grams of tissue were homogenized in low-ionic-strength buffer (50 mM NaC1, 4 mM EDTA, I mM EGTA, 2 mM DTT, 5 mg/1 leupeptin, 20 mM Tris, pH 7.5, 4~ The 10,000 g supernatant is denoted Extract A. The pellet was re-extracted with this buffer (Extract A-2), buffer "B" containing 40 mM KC1, I mM MgC12, I mM EGTA, I mM DTT, 0.2 mM PMSF, 0.05% Triton X-100, 20 mM Tris (Extract B+), buffer "B" minus Triton (Extract B-), and finally buffer B containing 80 mM KC1 and 30 mM MgC12 (Extract C). Total Ca 2+/CaM-dependent autocamtide-2 kinase activity in each extract is plotted. (B) Anion-exchange chromatography of Extract C on DEAE-Sephacel. Autocamtide-2 kinase activity was assayed in each fraction plus (solid circles) and minus (solid triangles) Ca 2+/CAM. Also plotted is absorbance (open squares) and the NaC1 gradient (dotted line).

II antibodies, we have identified the kinase from swine carotid arterial smooth muscle as an isozyme(s) of CaM-kinase II composed of 54- and 58-kDa subunits (Singer et al., 1993). Northern analysis and Western blotting with the &subunit-specific antibody (CK2Delta) are consistent with the interpretation that the 54- and 58-kDa subunits are forms of the ~- and ~/-subunits, respectively (Singer, H. A., unpublished data). Thus, this readily solubilized smooth muscle CaM-kinase II is similar to brain, heart, and liver CaMkinase II, which are large multimers composed of 54- to 60-kDa kinase subunits (Colbran and Soderling, 1990). Following repeated extraction with low-ionicstrength buffers, a substantial pool of Caa+/CaM dependent kinase activity (approximately half of the total) can be recovered by extracting a myofibril-rich 10,000 g pellet from swine carotid arterial smooth muscle with a buffer containing 30 mM MgC12 as the key ingredient (Fig. 6A). This extraction is similar to that which was shown to dissociate caldesmon and its associated CaM-kinase activity (Ikebe et al., 1990). The Ca 2+/CaM-dependent kinase activity in this extract resolves into two fractions on an anion-exchange column, with a large fraction flowing through, which is atypical for CaM-kinase II (Fig. 6B). Further analysis of these fractions by gel filtration chromatography and immunoblotting indicates that the salt-eluted activity

is similar to the readily solubilized CaM-kinase II in extracts A and B in that it has a native size of 550-650 kDa and is composed of 54- and 58-kDa subunits. In contrast, the fraction not retained by the anionexchange column is monomeric with an estimated size of 35 kDa. On the basis of a comparison of autophosphorylation kinetics, catalytic properties, and cross-reactivity with anti-CaM-kinase II antibodies, we have concluded that the low-molecular-weight kinase represents the catalytic/regulatory domain of a CaM-kinase II subunit, or a closely related kinase (Schworer et al., 1994). The physiological significance, if any, of this CaM-kinase II-related activity, or its relationship to the previously described CaM-kinase activity that co-purifies with caldesmon, is not clear at this time.

VI. POTENTIAL FUNCTIONS OF CALMODULIN-KINASE II IN SMOOTH MUSCLE Phosphorylation of caldesmon in vitro by CaMkinase II (or PKC) has been shown to interfere with caldesmon binding to F-actin, and results in a reversal of the caldesmon inhibitory effect on actin-activated myosin ATPase activity (Ngai and Walsh, 1987). This has led to speculation that these kinases may be in-

11. Calcium/Calmodulin-Dependent Protein Kinase II volved in regulating smooth muscle contractile activity by directly phosphorylating caldesmon. The fact that CaM-kinase II or a similar kinase activity copurifies with caldesmon and phosphorylates it in vitro to high stoichiometries is consistent with this possibility. However, this may be misleading since the specific sites phosphorylated in caldesmon by CaM-kinase II and PKC in vitro (Ikebe and Reardon, 1990b) have been shown to be different from the sites phosphorylated in intact arterial smooth muscle in response to addition of contractile stimuli (Adam et al., 1989, 1992). In these studies, the sites phosphorylated in caldesmon from intact tissues were the same as those phosphorylated in vitro by mitogen-activated protein kinase (MAPkinase). Thus, if CaM-kinase II is involved in regulating caldesmon phosphorylation and function in vivo, it may be indirect via effects on the MAP-kinase signaling pathway. Calponin is another smooth muscle thin filament-associated protein that inhibits actinactivated smooth muscle myosin ATPase in vitro (Takahashi et al., 1988). Like caldesmon, phosphorylation of calponin in vitro by CaM-kinase II interferes with its inhibitory actions on actin-activated myosin ATPase activity (Winder and Walsh, 1990). Because calponin has not yet been shown to be phosphorylated to a significant extent in intact smooth muscle (B~rany et al., 1991), the functional significance of the in vitro phosphorylations catalyzed by CaM-kinase II or PKC remains speculative. A strong case can be made for a physiologically important role for CaM-kinase II in phosphorylating and modulating the activity of MLCK in smooth muscle. MLCK is phosphorylated by CaM-kinase II in vitro (Hashimoto and Soderling, 1990; Ikebe and Reardon, 1990a) on a site near the calmodulin binding domain, resulting in decreased affinity of MLCK for calmodulin (Stull et al., 1990). Phosphorylation of this site in MLCK and a subsequent decrease in MLCK affinity for CaM occurs in vivo in cultured tracheal smooth muscle cells, in response to addition of contractile stimuli (Stull et al., 1990). Treatment of intact tracheal smooth muscle cells with the CaM-kinase II inhibitor KN-62 inhibits phosphorylation of MLCK and potentiates LC20 phosphorylation in response to Ca 2+-mobilizing stimuli (Tansey et al., 1992). Similar effects have been demonstrated in permeabilized cells using KN-62 or a peptide inhibitor corresponding to amino acids 281309 in the regulatory domain of CaM-kinase II (Tansey et al., 1994). The calcium dependence for phosphorylating MLCK in trachael cells has been reported to be half-maximal at a concentration of free intracellular Ca 2+ equal to 500 nM (Tansey et al., 1994), consistent with the in situ Ca 2+ dependence for activating CaMkinase II shown in Fig. 5B. Thus, CaM-kinase II ap-

15 1

pears to negatively modulate MLCK activity, resulting in the attenuation of LC20 phosphorylation, during activation of smooth muscle by contractile stimuli. CaM-kinase II may also be involved in control of smooth muscle contractile activity by regulating free intracellular [Ca2+]. CaM-kinase II and other multifunctional protein kinases have been reported to phosphorylate dihydropyridine receptors (Ca 2+ channels) in vitro (Jahn et al., 1988). Peptide inhibitors of CaMkinase II specifically block Ca2+-induced enhancement of L-type Ca 2+ current in isolated toad stomach smooth muscle cells (McCarron et al., 1992) or rabbit heart ventricular myocytes (Anderson et al., 1994). Thus CaM-kinase II has been implicated in the positive feedback control of voltage-regulated Ca 2+ channels. CaM-kinase II may also regulate Ca 2+ uptake into sarcoplasmic reticulum (SR). The cardiac SR Ca2+-ATPase (SERCA2) is under tonic inhibitory control by the SRassociated protein, phospholamban. Phosphorylation of phospholamban by cAMP-dependent protein kinase or CaM-kinase II relieves its inhibitory effect on SERCA2 activity, resulting in enhanced Ca 2+ uptake into SR (LePeuch et al., 1979). In addition, SERCA2 can be directly phosphorylated on a specific serine residue (Ser38) by an endogenous SR Ca2+/CaM-dependent kinase or exogenous CaM-kinase II, resulting in SERCA2 activation independently of phospholamban effects (Xu et al., 1993; Toyofuku et al., 1994). Other membrane channels, including anion channels from bovine tracheal epithelial cells that are phosphorylated and activated in vitro by CaM-kinase II (Fuller et al., 1994), may be physiologically relevant substrates for the kinase. Given the ubiquity of CaM-kinase II and the universal importance of Ca 2+ as a second messenger, it is not surprising that CaM-kinase II has been implicated in the control of other basic cellular processes that are not unique to smooth muscle. For example, there is increasing evidence that regulation of certain transcriptional responses by Caa+-dependent pathways is mediated by CaM-kinase II and/or CaM-kinase IV (Sun et al., 1994; Enslen and Soderling, 1994). Expression of a constitutively active fragment of CaM-kinase II acts to increase the rate of transcription of several genes (Kapiloff et al., 1991) by phosphorylating a transcription factor that binds to cAMP response elements (CREB) (Sheng et al., 1991; Sun et al., 1994). CaM-kinase II has also been implicated in the fundamental process of cell cycle control (Means, 1994). In nonmammalian models such as sea urchin (Baitinger et al., 1990) and frog (Lorca et al., 1993) oocytes, CaM-kinase II inhibitor peptides have been shown to block progression of mitosis. Transient expression of a truncated constitutively active form of CaM-kinase II in stable mammalian cell

15 2

HAROLD A. SINGER, S. THOMAS ABRAHAM, AND CHARLES M. SCHWORER

lines results in a complete arrest of cell cycle progression in the G2 phase (Planas-Silva and Means, 1992). Other data supporting proposed roles for CaM-kinase II in cell cycle control include an immunofluorescence study of several mammalian cell lines where CaMkinase II was shown to be localized in the nuclear matrix and nucleoli during interphase and in the mitotic apparatus during mitosis (Ohta et al., 1990). Studies establishing a structural basis for the localization of CaM-kinase II in the nucleus (Srinivasan et al., 1994) support the concept that CaM-kinase II is involved in the regulation of fundamental nuclear processes associated with gene transcription and cell division.

VII. SUMMARY

Even though CaM-kinase II is a ubiquitous multifunctional protein kinase controlled by a universal second messenger, relatively little is known about its specific cellular functions in tissues other than brain. This is true in smooth muscle, where there has been substantial speculation regarding roles for CaMkinase II in controlling or modulating contractile function. Studies using relatively specific pharmacological inhibitors of CaM-kinase II have hinted at the importance of the kinase in controlling contractile function by modulating availability of activator Ca 2+ and by phosphorylating and desensitizing MLCK. Molecular approaches have provided evidence that CaM-kinase II may be involved in controlling basic cellular processes such as gene transcription and cell growth. The unique autoregulatory properties of CaM-kinase II, which have been defined in vitro, have physiological implications that are only just beginning to be evaluated in vivo. Much additional work is required to assess the significance in vivo of specific autophosphorylation events that modify CaM-kinase II activator requirements or total activity. Biochemical and molecular approaches are starting to provide detailed knowledge of CaM-kinase II holoenzyme and subunit structure in smooth muscle. This information, coupled with a better basic understanding of how CaM-kinase II structure influences subcellular distribution, substrate specificity, and autoregulatory properties, should ultimately provide a basis for the development of specific tools and approaches aimed at establishing functional roles for the kinase.

References Abougou, J.-C., Hagiwara, M., Hachiya, T., Terasawa, M., Hidaka, H., and Hartshorne, D. J. (1989). FEBS Lett. 257, 408-410. Adam, L. P., Gapinski, C. J., and Hathaway, D. R. (1992). FEBS Lett. 302, 223-226.

Adam, L. P., Haeberle, J. R., and Hathaway, D. R. (1989). J. Biol. Chem. 264, 7698-7703. Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1982). J. Biol. Chem. 257, 8348-8355. Anderson, M. E., Braun, A. P., Schulman, H., and Premack, B. A. (1994). Circ. Res. 75, 854-861. Baitinger, C., Alderton, H., Poenie, M., Schulman, H., and Steinhardt, R. A. (1990). J. Cell Biol. 111, 1763-1773. B~irany, M., Rokolya, A., and B~ir~iny, K. (1991). FEBS Lett. 279, 6568. Bennett, M. K., and Kennedy, M. B. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 1794-1798. Benson, D. L., Isackson, P. J., Gail, C. M., and Jones, E. G. (1991). J. Neurosci. 11, 31-47. Bulleit, R. E., Bennett, M. K., Molfoy, S. S., Hurley, J. B., and Kennedy, M. B. (1988). Neuron 1, 63-72. Cho, K. O., Wall, J. B., Pugh, P. C., Ito, M., Mueller, S. A., and Kennedy, M. B. (1991). Neuron 7, 439-450. Chu, A., Sumbilla, C., Inesi, G., Jay, S. D., and Campbell, K. P. (1990). Biochemistry 29, 5899-5905. Cohen, M. E., Reinlib, L., Watson, A. J., Gorelick, F., Rys-Sikore, K., Tse, M., Rood, R. P., Czernik, A. J., Sharp, G. W., and Donowitz, M. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 8990-8994. Colbran, R. J. (1993). J. Biol. Chem. 268, 7163-7170. Colbran, R. J., and Soderling, T. R. (1990). Curr. Top. Cell. Regul. 31, 181-215. Colbran, R. J., Fong, Y.-L., Schworer, C. M., and Soderling, T. R. (1988). J. Biol. Chem. 263, 18145-18151. Cruzalegui, F. H., and Means, A. R. (1993). J. Biol. Chem. 268, 2617126178. Cruzalegui, F. H., Kapiloff, M. S., Morfin, J.-P., Kemp, B. E., Rosenfeld, M. G., and Means, A. R. (1992). Proc. Natl. Acad. Sci. U.S.A. 89, 12127-12131. DeRemer, M. F., Saeli, R. J., and Edelman, A. M. (1992). J. Biol. Chem. 267, 13460-13465. Edman, C. F., and Schulman, H. (1994). Biochim. Biophys. Acta 1221, 89-101. Enslen, H., and Soderling, T. R. (1994). J. Biol. Chem. 269, 2087220877. Erondu, N. E., and Kennedy, M. B. (1985). J. Neurosci. 5, 3270-3277. Frangakis, M. V., Ohmstede, C. A., and Sahyoun, N. (1991). J. Biol. Chem. 266, 11309-11316. Fukunaga, K., Rich, D. P., and Soderling, T. R. (1989). J. Biol. Chem. 264, 21830-21836. Fuller, C. M., Ismailov, I. I., Keeton, D. A., and Benos, D. J. (1994). J. Biol. Chem. 269, 26642-26650. Hanson, P. I., and Schulman, H. (1992). Annu. Rev. Biochem. 61,559601. Hanson, P. I., Kapiloff, M. S., and Lou, L. L. (1989). Neuron 3,59-70. Hanson, P. I., Meyer, T., Stryer, L., and Schulman, H. (1994). Neuron 12, 943-956. Hashimoto, Y., and Soderling, T. R. (1990). Arch. Biochem. Biophys. 278, 41-45. Hashimoto, Y., Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1987). J. Biol. Chem. 262, 8051-8055. Ikebe, M., and Reardon, S. (1990a). J. Biol. Chem. 265, 8975-8978. Ikebe, M., and Reardon, S. (1990b). J. Biol. Chem. 265, 17607-17612. Ikebe, M., Reardon, S., Scott-Woo, G. C., Zhou, Z., and Koda, Y. (1990). Biochemistry 29, 11242-11248. Jahn, H., Nastainczyk, W., Rohrkasten, A., Schneider, T., and Hofmann, F. (1988). Eur. J. Biochem. 178, 535-542. Jefferson, A. B., Travis, S. M., and Schulman, H. (1991). J. Biol. Chem. 266, 1484-1490. Kamm, K. E., and Stull, J. T. (1989). Annu. Rev. Physiol. 51, 299-313. Kanaseki, T., Ikeuchi, Y., Sugiura, H., and Yamauchi, T. (1991). J. Cell Biol. 115, 1049-1060.

11. Calcium/Calmodulin-Dependent Protein Kinase II Kapiloff, M. S., Mathis, J. M., Nelson, C. A., Lin, C. R., and Rosenfeld, M. G. (1991). Proc. Natl. Acad. Sci. U.S.A. 88, 3710-3714. Kelly, P. T., Shields, S., Conway, K., Yip, R., and Burgin, K. (1987). J. Neurochem. 49, 1927-1940. Lai, Y., Nairn, A. C., and Greengard, P. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 4253-4257. LePeuch, C. J., Haiech, J., and Demaille, J. G. (1979). Biochemistry 18, 5150-5157. Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A. F., Hanson, P., Schulman, H., and Rosenfeld, M. G. (1987). Proc. Natl. Acad. Sci. U.S.A. 84, 5962-5966. Lorca, T., Cruzalegui, F. H., Fesquet, D., Cavadore, J.-C., Mery, J., Means, A. R., and Dor6e, M. (1993). Nature (London) 366, 270273. Lou, L. L., Lloyd, S. J., and Schulman, H. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 9497-9501. MacNichol, M., Jefferson, A. B., and Schulman, H. (1990). J. Biol. Chem. 265, 18055-18058. Matovcik, L. M., Haimowitz, B., Goldenring, J. R., Czernik, A. J., and Gorelick, F. S. (1989). Am. J. Physiol. 244, C1029-C1036. Mayer, P., Mohlig, M., Schatz, H., and Pfeiffer, A. (1993). FEBS Lett. 333, 315-318. McCarron, J. G., McGeown, J. G., Reardon, S., Ikebe, M., Fay, F. S., and Walsh, J. V. (1992). Nature (London) 357, 74-77. Means, A. R. (1994). FEBS Lett. 347, 1-4. Means, A. R., Cruzalegui, F., LeMagueresse, B., Needleman, D. S., Slaughter, G. R., and Ono, T. (1991). Mol. Cell. Biol. 11, 39603971. Meyer, T., Hanson, P. I., Stryer, L., and Schulman, H. (1992). Science 256, 1199-1202. Miller, S. G., and Kennedy, M. B. (1986).Cell (Cambridge, Mass.) 44, 861-870. Mochizuki, H., Ito, T., and Hidaka, H. (1993). J. Biol. Chem. 268, 9143-9147. Mukherji, S., and Soderling, T. R. (1994). J. Biol. Chem. 269, 1374413747. Nairn, A. C., Bhagat, B., and Palfrey, C. (1985). Proc. Natl. Acad. Sci. U.S.A. 79, 7939-7943. Ngai, P. K., and Walsh, M. P. (1985). Biochem. J. 230, 695-707. Ngai, P. K., and Walsh, M. P. (1987). Biochem. J. 244, 417-425. Nghiem, P., Saati, S. M., Martens, C. L., Gardner, P., and Schulman, H. (1993). J. Biol. Chem. 268, 5471-5479. Ohsako, S., Nishida, Y., Ryo, H., and Yamauchi, T. (1993). J. Biol. Chem. 268, 2052-2062. Ohta, Y., Ohba, T., and Miyamoto, E. (1990). Proc. Natl. Acad. Sci. U.S.A. 87, 5341-5345. Papadopoulus, V., Brown, A. S., and Hall, P. F. (1990). Mol. Cell. Endocrinol. 74, 109-123. Patton, B. L., Miller, S. G., and Kennedy, M. B. (1990). J. Biol. Chem. 265, 11204-11212. Payne, M. E., Fong, Y.-L., Ono, T., Colbran, R. J., Kemp, B. E., Soderling, T. R., and Means, A. R. (1988). J. Biol. Chem. 263, 7190-7195. Pearson, R. B., Woodgett, J. R., Cohen, P., and Kemp, B. E. (1985). J. Biol. Chem. 260, 14471-14476. Picciotto, MI R., Czernik, A. J., and Nairn, A. C. (1993). ]. Biol. Chem. 268, 26512-26521.

153

Planas-Silva, M. D., and Means, A. R. (1992). EMBO J. 11, 507-517. Sahyoun, N., Levine, H., Bronson, D., Siegel-Greenstein, F., and Cuatrecasas, P. (1985). J. Biol. Chem. 260, 1230-1237. Schulman, H., and Hanson, P. I. (1993). Neurochem. Res. 18, 65-77. Schworer, C. M., Colbran, R. J., and Soderling, T. R. (1986). J. Biol. Chem. 261, 8581-8584. Schworer, C. M., Colbran, R. J., Keefer, J. R., and Soderling, T. R. (1988). J. Biol. Chem. 263, 13486-13489. Schworer, C. M., Rothblum, L. I., Thekkumkara, T., and Singer, H. A. (1993). J. Biol. Chem. 268, 14443-14449. Schworer, C. M., Sweeley, C. S., and Singer, H. A. (1994). FASEB J. 8, A393. Scott-Woo, G. C., and Walsh, M. P. (1988). Biochem. J. 255, 817-824. Scott-Woo, G. C., Sutherland, C., and Walsh, M. P. (1990). Biochem. ]. 268, 367-370. Sheng, M., Thompson, M. A., and Greenberg, M. E. (1991). Science 252, 1427-1430. Singer, H. A., Sweeley, C., and Schworer, C. M. (1993). Biophys. J. 64, A33. Srinivasan, M., Edman, C. F., and Schulman, H. (1994). J. Cell Biol. 126, 839-852. Stull, J. T., Hsu, L. C., Tansey, M. G., and Kamm, K. E. (1990). J. Biol. Chem. 265, 16683-16690. Sumi, M., Kiuchi, K., Ishikawa, T., Ishii, A., Hagiwara, M., Nagatsu, T., and Hidaka, H. (1991). Biochem. Biophys. Res. Commun. 181, 968-975. Sun, P., Enslen, H., Myung, P. S., and Maurer, R. A. (1994). Genes Dev. 8, 2527-2539. Takahashi, K., Hiwada, K., and Kokubu, T. (1988). Hypertension 11, 620-626. Tansey, M. G., Word, R. A., Hidaka, H., Singer, H. A., Schworer, C. M., Kamm, K. E., and Stull, J. T., (1992). J. Biol. Chem. 267, 12511-12516. Tansey, M. G., Luby-Phelps, K., Kamm, K. E., and Stull, J. T. (1994). J. Biol. Chem. 269, 9912-9920. Thiel, G., Czernik, A. J., Gorelik, F., Nairn, A. C., and Greengard, P. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 6337-6341. Tobimatsu, T., and Fujisawa, H. (1989). J. Biol. Chem. 264, 1790717912. Tobimatsu, T., Kameshita, I., and Fujisawa, H. (1988). J. Biol. Chem. 263, 16082-16086. Tokumitsu, H., Chijwa, T., Hagiwara, M., Mizutani, A., Terasawa, T., and Hidaka, H. (1990). J. Biol. Chem. 265, 4315-4320. Toyofuku, T., Kurzydlowski, K., Narayanan, N., and MacLennan, D. H. (1994). J. Biol. Chem. 269, 26492-26496. Winder, S. J., and Walsh, M. P. (1990). J. Biol. Chem. 265, 1014810155. Xu, A., Hawkins, C., and Narayanan, N. (1993). J. Biol. Chem. 268, 8394-8397. Yamagata, Y., Czernik, A. J., and Greengard, P. (1991). J. Biol. Chem. 266, 15391-15397. Yamauchi, T., Ohsaka, S., and Deguchi, T. (1989). J. Biol. Chem. 264, 19108-19116. Zhou, Z. L., and Ikebe, M. (1994). Biochem. J. 299, 489-495.