1Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208; and 2Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224
Submitted 3 December 2003 ; accepted in final form 29 January 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
calcium/calmodulin-dependent protein kinase II; cell migration; adenovirus; autophosphorylation; chemotaxis; platelet-derived growth factor
One regulatory protein implicated in VSM cell migration is Ca2+/calmodulin-dependent protein kinase II (CaMKII) (13, 15, 35). CaMKII is a multifunctional serine/threonine protein kinase with numerous alternatively spliced isoforms arising from four genes (,
,
, and
; see Refs. 10, 29). The isoforms have distinct tissue expression patterns, with the
- and
-isoforms located almost exclusively in brain tissue, whereas the
- and
-isoforms are found in both brain and peripheral tissues (10, 29). When free Ca2+ rises within the cell, Ca2+/calmodulin complexes bind to a conserved regulatory domain in CaMKII subunits, resulting in activation and substrate phosphorylation, including rapid autophosphorylation on Thr287 (Thr286 in the
-isoform) in the regulatory domain (9, 19). This autophosphorylation dramatically increases the binding affinity for calmodulin (16, 23) and renders the autoinhibitory domain inactive, providing the kinase with 5070% autonomous activity in the absence of Ca2+/calmodulin (9). Functionally, this provides the kinase with the capacity to respond to Ca2+ transients in a frequency-dependent manner (6, 8). In addition, phosphorylation of this residue has been shown to be important in targeting the enzyme (26), facilitating the interaction of CaMKII and its substrates (34).
CaMKII is expressed in vivo as a large holoenzyme composed of up to 12 individual kinase subunits (10). Formation of the holoenzyme is dependent on a conserved COOH-terminal association domain (10) and has been shown to be important for autophosphorylation that results from an intersubunit, intraholoenzyme reaction (4). Because the holoenzyme structure can be heteromultimeric (29), a number of investigators are beginning to study how subunit composition affects holoenzyme targeting (2, 5, 27, 30). For example, a short, variable domain conserved in a number of subunits was demonstrated to contain a nuclear localization sequence capable of targeting CaMKII holoenzymes to the nucleus (25). In addition, the -isoform of CaMKII has been shown to contain domains that provide targeting to the cytoskeleton and F-actin (21, 30). Such findings emphasize the importance of defining the CaMKII isoforms expressed in a specific cell type and considering structural features that may result in distinct protein-protein interactions and unique function.
Previous studies using cultured VSM cells implicate a regulatory role for CaMKII in PDGF-stimulated chemotactic cell migration (13, 15). These studies relied on chemical inhibition of CaMKII (with KN-62 or KN-93) or molecular manipulation of CaMKII activity by expression of a constitutively active CaMKII, an isoform not expressed in VSM (29, 35). We (20) previously reported that the
gene is the main isoform expressed in cultured VSM, with the
2 splice variant (also called
c in some studies) being the major CaMKII product expressed, along with minor levels of
3 (
B) detectable by RT-PCR and Western blot analysis. In the present study, we inhibited endogenous CaMKII
2 function by using an adenoviral infection approach to introduce a kinase-negative mutant of CaMKII
2 that was demonstrated to act as a dominant negative with respect to CaMKII activity. Whereas KN-93 was shown to inhibit PDGF-stimulated VSM cell migration, overexpression of the kinase-negative CaMKII
2 potentiated PDGF-induced migration. Furthermore, expression of a constitutively active
2-isoform inhibited VSM cell migration. The results of these studies necessitate a careful reexamination of the mechanisms by which CaMKII regulates VSM cell migration, cognizant of potential isoform-specific functions and/or functions uniquely related to its autophosphorylation state.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. VSM cells were dispersed from thoracic aortas of 200- to 300-g Sprague-Dawley rats as previously described (7). Cells were cultured in combined DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT) at 37°C and 5% CO2. Cells from passages 310 were used for experiments. Cells were grown to 70% confluence before infection with adenoviral constructs.
Cloning and adenovirus generation.
Mutations of CaMKII2 were engineered using the Transformer Site Directed Mutagenesis kit (Clontech, Palo Alto, CA). Kinase-negative CaMKII
2 was generated by replacing Lys43 with an alanine. Amino acid substitution of Thr287 to aspartic acid generated a constitutively active CaMKII
2 mutant. M. T. Crow provided adenoviral stocks encoding kinase-negative (Ad-KN:
2) and constitutively active (Ad-CA:
2) CaMKII. Adenovirus encoding
-galactosidase (Ad-
Gal) was a gift from R. S. Keller. RT-PCR was used (Titan one-tube RT-PCR kit; Roche, Indianapolis, IN) to clone PLB from isolated rat brain RNA. Primers were designed according to the rat PLB cDNA sequence (accession no. L03382.1): upstream, 5'-GAGAGGATCC(814)ATGGAAAAAGTCCAATACCT(833)-3', and downstream, 5'-GAGAACTCGAG(972)TCACAGAAGCATCACAATG(953)-3'. The amplified product containing the entire coding region was then cloned into pCMV-tag2B (Invitrogen, La Jolla, CA) and subcloned by digesting Flag:PLB cDNA with SnaB1 and Xho1 (New England Biolabs, Beverly, MA) followed by ligation into a pShuttle vector (provided by Dr. Peter A. Vincent, Center for Cardiovascular Sciences, Albany Medical College, Albany, NY) for virus generation. pShuttle:PLB was linearized and introduced by electroporation into BJ5183 cells containing the pAdEasy backbone for recombination. The resulting recombined vector was linearized by Pac1 digestion and transfected into HEK-293 cells for viral packaging.
All adenovirus stocks were propagated by adding small amounts of virus to HEK-293 cells. When cells were 50% lysed, cells and media were collected, subjected to freeze-thaw cycles three times, and then aliquoted and stored at 80°C. Titer assays were performed according to the method of O'Carroll et al. (14). All assays were performed using
-galactosidase as an adenoviral control at matching multiplicity of infection (MOI).
Cell lysates, immunoprecipitation, and immunoblotting. Thirty minutes before experimentation, growth medium was removed from VSM cells and replaced with Hanks' balanced salt solution (HBSS) supplemented with Ca2+/Mg2+ and 10 mM HEPES, pH 7.4. Reactions were stopped by removing HBSS, transferring the dishes to ice, and adding RIPA lysis buffer (4°C, 10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.2 U/ml aprotinin) at 0.3 ml/60-mm dish. The lysates and cells were collected into 1.5-ml tubes and then centrifuged at 14,000 rpm at 4°C for 10 min, and the supernatant was collected for Western blot analysis or storage at 20°C.
For immunoprecipitation of PLB, 60-mm dishes infected and treated with an appropriate stimulus were lysed in 500 µl of immunoprecipitate (IP) buffer (500 mM Tris, pH 7.4, 50 mM NaF, 0.1 mM NaVO4, and 0.5% NP-40). Dishes were scraped, and lysates were cleared by centrifugation at 14,000 rpm at 4°C for 10 min. Monoclonal anti-Flag antibody (2 µg) and 40 µl of protein A beads were added to each IP (Pierce, Richmond, CA). After immunoprecipitation overnight at 4°C, they were washed three times the next day in lysis buffer followed by the addition of SDS sample buffer.
Lysates and IPs were resolved by 9 or 15% SDS-PAGE gel and transferred to nitrocellulose. The membranes were blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% nonfat dry milk. After blocking, the membranes were incubated in primary antibody for 1 h at room temperature, washed three times with TBST, and incubated with horseradish peroxidase-conjugated secondary antibody (1:1,500 dilution; Amersham) for an additional hour at room temperature, then washed three times with TBST. Membranes were developed using chemiluminescence substrate (Amersham) and exposed to Hyperfilm ECL (Amersham Biosciences, Piscataway, NJ). PLB phosphorylation was quantified by densitometric analysis of the immunoblot enhanced chemiluminescence signals with the use of ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Additional antibody dilutions used included CaMKII2 antibody at 1:1,000, polyclonal antibody for phosphorylated CaMKII at 1:500, polyclonal HA and monoclonal Flag antibodies at 1:1,000, and phosphorylated PT17 at 1:5,000.
CaMKII activity assay. Thirty-six to forty-eight hours after infection with adenoviral constructs, 300 µl of lysis buffer (50 mM MOPS, pH 8.6, 100 mM Na4P2O7, 100 mM NaF, 250 mM NaCl, 2 mM Na3VO4, 3 mM EGTA, and 1% NP-40) was added to each 60-mm dish. Lysates were scraped and centrifuged at 14,000 rpm for 10 min at 4°C. Total CaMKII activity in the presence of saturating Ca2+ and calmodulin, as well as autonomous activity, assayed without added activators, was determined as described previously using autocamtide II, a specific peptide substrate (1).
Cell migration assays. Cultured VSM cells were grown in medium containing 10% FBS (as described in Cell culture) for 3648 h after infection with adenoviral constructs. All 60-mm dishes were washed once in HBSS, trypsinized, and seeded at 150,000 cells/modified Boyden chamber (PET track-etched, 8-µM pores, 12-well format; Becton Dickinson, San Diego, CA) coated with 3 µg/ml fibronectin (Sigma). Chambers were prepared by adding to the lower chambers medium containing either DMEM with 0.4% FBS (control) or 10 ng/ml PDGF-BB in DMEM with 0.4% FBS. Cells were permitted to migrate for 4 h. The tops of the membranes were swabbed to remove cells, and then cells on the bottom surface of the membrane were fixed for 30 min in PBS containing 4% paraformaldehyde (pH 7.4). Cells were stained for 10 min in Coomassie blue. Filters were removed from the chamber and mounted onto microscope slides on which four identically located fields per membrane were averaged for quantification of cell number.
Adhesion assay. Thirty-six to forty-eight hours after infection with adenoviral constructs, the ability of the cells to adhere to a fibronectin-coated surface (10 µg/ml; Sigma) was assayed as previously described (3).
Statistical evaluations and comparisons. All data are expressed as means ± SE. Mean values of groups were compared by ANOVA with post hoc comparisons using the Newman-Keuls test. For all comparisons, P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
To determine the effects of expressing these constructs on CaMKII activity, in vitro kinase assays (1) were performed using lysates of control and infected cells (Fig. 2). CaMKII activity was assayed both with and without added Ca2+ and calmodulin to obtain total and autonomous kinase activity, respectively. Infection of cells with 10 MOI of Ad-KN:2, which produced approximately fivefold more mutant subunits relative to endogenous subunits (Fig. 1B), decreased total CaMKII specific activity in VSM cell lysates by
50%. Basal autonomous activity was not detected in cells infected with 10 MOI of Ad-KN:
2 (Fig. 2). Conversely, comparable increases in the expression levels of the constitutively active
2 mutant after infection with 10 MOI of Ad-CA:
2 significantly increased both autonomous and total CaMKII activity in VSM cell lysates (Fig. 2). The level of autonomous activity observed with this MOI of active
2 is comparable to the total Ca2+/calmodulin-stimulated activity in control cells. Infection with Ad-
Gal did not significantly alter CaMKII specific activity.
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Analysis of CaMKII2 phosphorylation on Thr287, however, indicated that overexpression of the kinase-negative mutant at this level did not block the highly cooperative (17, 23) autophosphorylation reactions and, in fact, that it served as a substrate for these reactions (Fig. 4). Because the kinase-negative subunit is unable to catalyze autophosphorylation, and because these reactions have been demonstrated to be intersubunit within the multimeric holoenzyme (4), these data indicate that the mutant subunit integrates into holoenzymes with endogenous
2-subunits and suggest proper targeting of the mutant subunits. Conversely, in cells overexpressing the constitutively active mutant, endogenous CaMKII
2 Thr287 autophosphorylation was inhibited. This result has a number of potential explanations, including inactivating autophosphorylations catalyzed by the mutant on Thr306/307 in the calmodulin binding domain (19) and/or upregulated phosphatase activities enhancing reversal of the autophosphorylation events. Regardless of the mechanisms involved, overexpressing these mutants in Thr287 autophosphorylation could be an important consideration in interpreting the functional effects of these mutants, as in the present study and in previously published experiments in which acute (13) or stable (15, 35) overexpression approaches were used.
Several previous reports have indicated a role for CaMKII in VSM cell migration (13, 15, 35). A consistent finding confirmed in the present study is that selective pharmacological inhibitors of CaMKII (KN-62 or KN-93) inhibit migration of VSM cells toward chemotactic stimuli (13, 15). These agents act by interfering with Ca2+/calmodulin binding and subsequent activation, including autophosphorylation reactions (Fig. 4A). In the present study, however, introduction of the kinase-negative CaMKII2 mutant into VSM cells enhanced migration toward PDGF (Fig. 6) or conditioned medium. We currently have no definitive explanation for this discrepancy in results with the use of the different approaches. Potential explanations are that KN-93 exerts nonspecific effects on VSM cell migration or that the kinase-negative construct enhances migration through an effect unrelated to CaMKII per se. Another, more interesting explanation that is consistent with both the pharmacological and molecular approaches is that the requirement for CaMKII activation in cell migration necessitates autophosphorylation.
A requirement for CaMKII autophosphorylation in cell migration is also consistent with the results obtained with overexpression of the constitutively active CaMKII2 mutant. In these experiments, infection with constitutively active T287D mutant was titrated to achieve a level of expression that resulted in autonomous activity that was quantitatively comparable to the maximum levels of endogenous Ca2+/calmodulin-stimulated activity expressed in normal cells (Fig. 2). As discussed above, however, the effect of overexpressing this mutant on autophosphorylation of endogenous subunits was inhibitory, and the net effect on VSM cell migration also was inhibitory.
Similar studies using overexpressed constitutively active CaMKII mutants have been published, but with different conclusions. In general, the prior studies found enhanced VSM cell migration (35) or rescue of KN-62 or KN-93 inhibition of PDGF-stimulated cell migration (13, 15). Differences between the current and prior studies may factor into why essentially opposite results were found in our study. In many of the previous studies, the constitutively active construct was stably expressed (15, 35). It could be expected that this approach would result in significant adaptive changes, including the induction of phosphatase activity, as indicated by experiments evaluating CaMKII in VSM cell migration in response to wounding (35). With the use of the more acute adenoviral infection approach, as in the present study, there is less time for such adaptive changes to occur. In addition, in those prior experiments in which constitutively active CaMKII constructs were overexpressed using an adenoviral delivery system, the constructs were mutants of the CaMKII isoform (13) and contained a second charged residue substitution (V287D) in addition to the charge substitution at the autophosphorylation site in the
-subunit (T286D). To the extent that unique sequences in the
2-isoform and/or autophosphorylation levels may be important in holoenzyme targeting, the more densely charged, constitutively active
-subunit may act differently from the constitutively active
2-construct used in the present study. A potential requirement for CaMKII autophosphorylation in VSM cell migration (discussed above) might be satisfied by the more densely charged mutant and not by the
2-mutant, which also inhibited endogenous subunit autophosphorylation. Finally, an important limitation of any experiment expressing a constitutively active multifunctional serine/threonine kinase is persistent, unregulated phosphorylation of normal substrates as well as recruitment of nonphysiological substrates.
With the use of new molecular approaches, the present study indicates an important role for CaMKII2 in regulating cultured VSM cell migration. If interpreted strictly in the context of CaMKII
2 activity toward substrates other than itself, the results are not consistent with the effects of chemical inhibitors of the kinase. Alternatively, an inclusive interpretation of the results could point to a requirement for CaMKII autophosphorylation in VSM cell chemotactic migration. Although it is speculation at this point, this possibility is consistent with an increasing number of results in other systems indicating the importance of autophosphorylation events in directing CaMKII holoenzyme targeting (2) and enzymatic activity (34). The ultimate cellular mechanisms underlying CaMKII actions on cell migration are also not yet known but could be related to effects of the kinase on the stability and turnover of focal adhesions (32) or on the activity of the contractile apparatus, perhaps by inhibiting myosin light chain kinase activity (28). The present results indicate the need for additional studies to clarify the mechanisms by which CaMKII
2 regulates VSM cell migration and whether these results can be extrapolated to other cell systems.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Bayer KU and Schulman H. Regulation of signal transduction by protein targeting: the case for CaMKII. Biochem Biophys Res Commun 289: 917923, 2001.[CrossRef][ISI][Medline]
3. Bilato C, Curto KA, Monticone RE, Pauly RR, White AJ, and Crow MT. The inhibition of vascular smooth muscle cell migration by peptide and antibody antagonists of the alphavbeta3 integrin complex is reversed by activated calcium/calmodulin-dependent protein kinase II. J Clin Invest 100: 693704, 1997.
4. Bradshaw JM, Hudmon A, and Schulman H. Chemical quenched flow kinetic studies indicate an intraholoenzyme autophosphorylation mechanism for Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 277: 2099120998, 2002.
5. Caran N, Johnson LD, Jenkins KJ, and Tombes RM. Cytosolic targeting domains of gamma and delta calmodulin-dependent protein kinase II. J Biol Chem 276: 4251442519, 2001.
6. Dosemeci A and Albers RW. A mechanism for synaptic frequency detection through autophosphorylation of CaM kinase II. Biophys J 70: 24932501, 1996.[Abstract]
7. Geisterfer AA, Peach MJ, and Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res 62: 749756, 1988.[Abstract]
8. Hanson PI, Meyer T, Stryer L, and Schulman H. Dual role of calmodulin in autophosphorylation of multifunctional CaM kinase may underlie decoding of calcium signals. Neuron 12: 943956, 1994.[ISI][Medline]
9. Hashimoto Y, Schworer CM, Colbran RJ, and Soderling TR. Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. Effects on total and Ca2+-independent activities and kinetic parameters. J Biol Chem 262: 80518055, 1987.
10. Hudmon A and Schulman H. Structure-function of the multifunctional Ca2+/calmodulin-dependent protein kinase II. Biochem J 364: 593611, 2002.[CrossRef][ISI][Medline]
11. Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. Targeted inhibition of Ca2+/calmodulin-dependent protein kinase II in cardiac longitudinal sarcoplasmic reticulum results in decreased phospholamban phosphorylation at threonine 17. J Biol Chem 278: 2506325071, 2003.
12. Li L, Chu G, Kranias EG, and Bers DM. Cardiac myocyte calcium transport in phospholamban knockout mouse: relaxation and endogenous CaMKII effects. Am J Physiol Heart Circ Physiol 274: H1335H1347, 1998.
13. Lundberg MS, Curto KA, Bilato C, Monticone RE, and Crow MT. Regulation of vascular smooth muscle migration by mitogen-activated protein kinase and calcium/calmodulin-dependent protein kinase II signaling pathways. J Mol Cell Cardiol 30: 23772389, 1998.[CrossRef][ISI][Medline]
14. O'Carroll SJ, Hall AR, Myers CJ, Braithwaite AW, and Dix BR. Quantifying adenoviral titers by spectrophotometry. Biotechniques 28: 40810, 412, 2000.[ISI][Medline]
15. Pauly RR, Bilato C, Sollott SJ, Monticone R, Kelly PT, Lakatta EG, and Crow MT. Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration. Circulation 91: 11071115, 1995.
16. Putkey JA and Waxham MN. A peptide model for calmodulin trapping by calcium/calmodulin-dependent protein kinase II. J Biol Chem 271: 2961929623, 1996.
17. Rich RC and Schulman H. Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 273: 2842428429, 1998.
18. Schwartz SM, Campbell GR, and Campbell JH. Replication of smooth muscle cells in vascular disease. Circ Res 58: 427444, 1986.[Abstract]
19. Schworer CM, Colbran RJ, Keefer JR, and Soderling TR. Ca2+/calmodulin-dependent protein kinase II. Identification of a regulatory autophosphorylation site adjacent to the inhibitory and calmodulin-binding domains. J Biol Chem 263: 1348613489, 1988.
20. Schworer CM, Rothblum LI, Thekkumkara TJ, and Singer HA. Identification of novel isoforms of the delta subunit of Ca2+/calmodulin-dependent protein kinase II. Differential expression in rat brain and aorta. J Biol Chem 268: 1444314449, 1993.
21. Shen K, Teruel MN, Subramanian K, and Meyer T. CaMKIIbeta functions as an F-actin targeting module that localizes CaMKIIalpha/beta heterooligomers to dendritic spines. Neuron 21: 593606, 1998.[ISI][Medline]
22. Simmerman HK, Collins JH, Theibert JL, Wegener AD, and Jones LR. Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains. J Biol Chem 261: 1333313341, 1986.
23. Singla SI, Hudmon A, Goldberg JM, Smith JL, and Schulman H. Molecular characterization of calmodulin trapping by calcium/calmodulin-dependent protein kinase II. J Biol Chem 276: 2935329360, 2001.
24. Smith MK, Colbran RJ, Brickey DA, and Soderling TR. Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II. Role of His282 and multiple basic residues. J Biol Chem 267: 17611768, 1992.
25. Srinivasan M, Edman CF, and Schulman H. Alternative splicing introduces a nuclear localization signal that targets multifunctional CaM kinase to the nucleus. J Cell Biol 126: 839852, 1994.[Abstract]
26. Strack S and Colbran RJ. Autophosphorylation-dependent targeting of calcium/ calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl-d-aspartate receptor. J Biol Chem 273: 2068920692, 1998.
27. Takeuchi Y, Yamamoto H, Fukunaga K, Miyakawa T, and Miyamoto E. Identification of the isoforms of Ca2+/calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization. J Neurochem 74: 25572567, 2000.[CrossRef][ISI][Medline]
28. Tansey MG, Luby-Phelps K, Kamm KE, and Stull JT. Ca2+-dependent phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain phosphorylation within smooth muscle cells. J Biol Chem 269: 99129920, 1994.
29. Tobimatsu T and Fujisawa H. Tissue-specific expression of four types of rat calmodulin-dependent protein kinase II mRNAs. J Biol Chem 264: 1790717912, 1989.
30. Urushihara M and Yamauchi T. Role of beta isoform-specific insertions of Ca2+/calmodulin-dependent protein kinase II. Eur J Biochem 268: 48024808, 2001.
31. Van Riper DA, Schworer CM, and Singer HA. Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle. Mol Cell Biochem 213: 8392, 2000.[CrossRef][ISI][Medline]
32. Wehrle-Haller B and Imhof BA. Actin, microtubules and focal adhesion dynamics during cell migration. Int J Biochem Cell Biol 35: 3950, 2003.[CrossRef][ISI][Medline]
33. Welt FG and Rogers C. Inflammation and restenosis in the stent era. Arterioscler Thromb Vasc Biol 22: 17691776, 2002.
34. Yasugawa S, Fukunaga K, Yamamoto H, Miyakawa T, and Miyamoto E. Autophosphorylation of Ca2+/calmodulin-dependent protein kinase II: effects on interaction between enzyme and substrate. Jpn J Pharmacol 55: 263274, 1991.[ISI][Medline]
35. Zhang S, Yang Y, Kone BC, Allen JC, and Kahn AM. Insulin-stimulated cyclic guanosine monophosphate inhibits vascular smooth muscle cell migration by inhibiting Ca/calmodulin-dependent protein kinase II. Circulation 107: 15391544, 2003.
36. Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J Jr, Bers DM, and Brown JH. The deltaC isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ Res 92: 912919, 2003.