Proc. Nati. Acad. Sci. USA Vol. 87, pp. 1273-1277, February 1990 Biochemistry
Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates Ca2+/calmodulin-independent activity (bacterial expression/autophosphorylation/pseudosubstrate)
M. NEAL WAXHAM*, JAROSLAW ARONOWSKI*, SARA A. WESTGATE*, AND PAUL T. KELLYt Departments of *Neurology and tNeurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston TX 77225
Communicated by Philip Siekevitz, November 13, 1989
ABSTRACT
We have examined the role of Thr-286 auto-
phosphorylation in the autoregulation of Ca2+/calmodulindependent protein kinase U. Using site-directed mutagenesis, we have substituted alanine or serine for Thr-286, or isoleucine for Arg-283, in the 50-kDa subunit of the kinase and expressed each protein in bacteria. Activation and autophosphorylation of all four enzymes were stringently dependent on Ca2+/ calmodulin, indicating that neither Arg-283 nor Thr-286 is an absolute requirement for the pseudosubstrate inhibition of the enzyme. Autophosphorylation of the Ile-283 or Ala-2%6 enzyme generated little, if any, Ca2+/calmodulin-independent kinase activity, unlike the parent (Thr-286) or Ser-286 enzyme. The enzymes expressed in bacteria are predominantly monomeric, indicating that the generation of Ca2+/calmodulin-independent activity does not require the cooperative interactions of subunits normally present in the brain holoenzyme.
Ca2+/calmodulin-dependent protein kinase II (CaM-KII) is the predominant protein kinase in mammalian brain and is a major protein component of neuronal postsynaptic densities (1, 2). When activated, CaM-KII phosphorylates a variety of substrates, thereby potentially modulating enzymes and proteins important in neuronal function and plasticity. A dynamic feature of CaM-KII is its autoregulatory property in which Ca2+/CaM-dependent autophosphorylation converts the kinase into a catalytic entity that no longer requires Ca2+/CaM for activity (3-11). This self-regulatory property, together with the subcellular localization of CaM-KII at postsynaptic densities, has led to hypotheses that implicate CaM-KII as a key element in mechanisms encoding the history of synaptic activity (12), a primary event in learning and memory. CaM-KII is a member of a growing family of enzymes autoregulated by pseudosubstrate inhibitory sequences (13). Pseudosubstrates possess substrate-like properties that enable them to bind at or near an enzyme's catalytic and/or substrate-binding domain, thereby inhibiting activity (14-19). The regulation of CaM-KII has been documented through the use of synthetic peptide inhibitors that contain the CaMbinding domain of the enzyme (14-19). Kelly et al. (14) have shown that the pseudosubstrate domain includes an autophosphorylation site (Arg-Gln-Glu-Thr, residues 283-286) on the N-terminal side of the CaM-binding domain. The recent sequencing of autophosphorylated peptides has suggested that Thr-286 in the pseudosubstrate domain is autophosphorylated during generation of Ca2+/CaM-independent kinase activity (20-22). The cloning (23-25) and expression of CaM-KII in bacteria (26) have allowed us to examine the effects of amino acid mutations at Arg-283 and Thr-286 on the generation of the Ca2+/CaM-independent form of CaM-KIT. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Coincidentally, we have also addressed the role of Arg-283 and Thr-286 in the pseudosubstrate regulation of CaM-KI.
MATERIALS AND METHODS Mutagenesis and Isolation of Bacterially Expressed CaMKU. We previously reported (26) the expression in bacteria of the 50-kDa subunit of CaM-KII using the expression vector pK223-2. The original construct contained a cDNA encoding three additional amino acids at the N terminus of the expressed protein. We have reconstructed the 50-kDa cDNA so that the expressed protein now starts at the initiating methionine residue of native CaM-K11. This modification increased the amount of expressed 50-kDa subunit =5-fold. Growth of bacteria and induction of CaM-K11 protein have been described (26). Hypotonic lysates of bacterial protoplasts were clarified by centrifugation at 50,000 X g for 20 min at 40C to obtain soluble and particulate fractions. Soluble and particulate fractions were assayed for Ca2"/ CaM-dependent kinase activity using a synthetic peptide as substrate (see below); 90% Ca2+/
A) Ala-286 10 Co 0
T-
V
SUBSTRATE (NTP, uM)
1/S
C) Ala-286
Inm
lV
D) Thr-286
30
3
25
2,
!5
K m 1.8 uM Vma = 0.20 uMoV
20
21!0
min/mg
-15
-1 5
-
0)
V
V
2
10
5 -0.2 -0.1
0.0
0.1
0.2
1/S
0.3
0.4
0.5
01 -0.2 -0.1
Km7.1 uM Vmax = 0.45 umoVmirnmg 0.0
0.1
0.2
0.3
0.4
0.5
1/S
FIG. 2. Kinetic analysis of bacterially expressed 50-kDa subunits at various concentrations of synthetic peptide (NTP). Phosphorylation reactions were carried out with the Ca2+/CaM-dependent (e) or Ca2+/CaM-independent (0) forms of the different enzymes; reactions were performed in duplicate and each mixture contained 50 ng of 50-kDa subunits. (A) Peptide phosphorylation by the Ala-286-substituted enzyme. (B-D) Double-reciprocal plots of peptide phosphorylation with Ser-286, Ala-286, or Thr-286 enzyme, respectively. The apparent Km and Vmax for each enzyme are included in the appropriate panel.
1276
Biochemistry: Waxham et al. 100. 80
A
Blue-Dex.
-
0,0 Arg-283 AA Ile-283
A.
-
c
8 40
A
,
/
60
Proc. Natl. Acad. Sci. USA 87 (1990)
CO
x
A Holoenzyme
* Thr-286 o Ala-286
'T5 0.2
C,,
20
Cyto-C
+ +
0.6 0.4
-.
I//-
-
I');o
BSA
-
0.0 ~ ~ ~ ~ ~
~
~
~
~
~
~
~
~~~~~-
10
AI.
0
100. 0
80 .
0.
60 .
60
30
90
120
150
*
Arg-283
A' Ile-283
40 .
c
(D Q
20 . 0. 20
.
40
140
60
80 100 Time (sec)
120
30
35
FIG. 4. Gel filtration analysis of rat forebrain CaM-KII and bacterially expressed CaM-KlI. Each enzyme was purified by phosphocellulose chromatography and analyzed by gel filtration. Fractions (1 ml) were collected and 20 ,ul was analyzed for Ca2+/ CaM-dependent protein kinase activity; A, rat brain holoenzyme; *, bacterially expressed Thr-286 enzyme; o, Ala-286 enzyme. Elution peaks for the molecular mass standards Blue dextran 2000 (2000 kDa), bovine serum albumin (BSA, 67 kDa) and cytochrome c (12.3 kDa) are indicated.
..
. _.
25 20 Volume (mL)
210
B
0X c
180
15
140
FIG. 3. Kinetic comparisons of bacterially expressed Arg-283 (circles) and Ile-283 (triangles) enzymes. (A) Equivalent amounts of each 50-kDa subunit (100 ng) were examined for Ca2+/CaMdependent phosphorylation of 30 ,uM syntide (cpm x 10-3, open symbols) or 50-kDa autophosphorylation (cpm x 10-2, filled symbols); reactions varied from 20 to 180 sec. (B) The generation of Ca2+/CaM-independent activity was analyzed for equivalent amounts of Arg-283 or Ile-283 enzyme; each enzyme was autophosphorylated for 30, 60, or 120 sec and then allowed to phosphorylate substrate in the presence of Ca2+ or EGTA. Independent activity (%) was calculated from ratios of substrate phosphorylation in EGTA vs. c2+.
Ca2~
Arg-283 contributes to the efficient generation of Ca2+/ CaM-independent activity. Gel filtration analyses (Fig. 4) showed that both the Thr286 and Ala-286 enzymes were eluted between bovine serum albumin (67 kDa) and cytochrome c (12.3 kDa), indicating that the enzymes expressed in bacteria are predominantly monomeric. Small amounts of the Thr-286 enzyme were eluted ahead ofbovine serum albumin, suggesting that a small proportion of this preparation may contain oligomeric forms of the enzyme. None of these apparent oligomers was present in the Ala-286 preparations. CaM-KII isolated from rat forebrain was eluted just after Blue dextran (2000 kDa) but well before bovine serum albumin, consistent with previous reports that the brain enzyme is multimeric (1, 27). Many kinases are regulated by pseudosubstrate inhibitory domains (13-18). While CaM-KII is among the enzymes activated by Ca2+/CaM, it contains a bona fide phosphorylation sequence within its inhibitory domain. Moreover, although many kinases undergo autophosphorylation, subsequent functional changes are much less dramatic than those displayed by CaM-KII. The pseudosubstrate domain of CaMKII appeared to require the consensus phosphorylation se-
quence Arg-Gln-Glu-Thr286 (14, 19), whose phosphorylation resulted in the generation of Ca2+/CaM-independent activity. By coupling the pseudosubstrate and CaM-binding domains, CaM-KII regulates the efficiency of Thr-286 phosphorylation; in the absence of Ca2+/CaM the former is an inhibitor, while in their presence it becomes a substrate. This hypothesis was initially supported by studies which showed that the intact sequence Arg-Gln-Glu-Thr in longer pseudosubstrate peptides was required for efficient inhibition (IC50 = 2 ,uM) of the Ca2+/CaM-independent form of CaMKII (14, 19). The latter studies also implied that disruption of critical determinants in this sequence, namely Arg-283 and Thr-286, may eliminate pseudosubstrate inhibition. We have shown that arginine at position 283 and threonine at position 286 are not absolute requirements for a functional pseudosubstrate inhibitory domain. The Ile-283, Ala-286, and Ser-286 enzymes each remained completely dependent on Ca2+/CaM for activity and followed similar CaM-activation kinetics when compared to the parent enzyme. Thus, the precise function and location of the pseudosubstrate domain in the native kinase are clearly more complex than studies with synthetic peptides had predicted. Further site-directed mutagenesis studies may reveal the critical amino acids responsible for the pseudosubstrate inhibition in native CaM-KII. CaM-KII purified from rat forebrain is a multisubunit holoenzyme containing two distinct protein subunits (50 and 60 kDa). It is clear that the kinase's ability to convert to a Ca2+/CaM-independent enzyme does not require both subunits (26), and the gel filtration data presented here indicate that the generation of autonomous catalytic activity can reside in a single monomeric subunit. A recent report (30) describing the transient expression of the 50-kDa subunit in eukaryotic cells and subsequent analysis of whole-cell extracts also determined that substitution of alanine for Thr-286 produced an enzyme that displayed very little Ca2+/CaMindependent activity after prior autophosphorylation. Interestingly, the enzyme expressed in eukaryotic cells is a holoenzyme of 6-10 subunits. We have reached similar conclusions with bacterially expressed enzymes and have shown that monomers of the 50-kDa subunit exhibit Ca2+/ CaM-independent activity after autophosphorylation. This indicates that cooperativity between subunits is not required for generation of Ca2+/CaM-independent activity.
Biochemistry: Waxham et al. We thank Allison Stock for technical assistance, Vikram Dabhi for help with the gel filtration studies, and Randall Kincaid for CaMSepharose. This work was supported by U.S. Public Health Service Grant NS26086. 1. Kennedy, M. B., Bennett, M. K. & Erondu, E. (1983) Proc. Natl. Acad. Sci. USA 80, 7357-7361. 2. Kelly, P. T., McGuinness, T. & Greengard, P. (1984) Proc. NatI. Acad. Sci. USA 81, 945-949. 3. Shields, S. M., Vernon, P. J. & Kelly, P. T. (1984) J. Neurochem. 43, 1599-1609. 4. Kelly, P. T., Yip, R., Shields, S. M. & Hay, M. (1985) J. Neurochem. 45, 1620-1634. 5. Saitoh, T. & Schwarz, J. H. (1985) J. Cell Biol. 100, 835-842. 6. Lai, Y., Nairn, A. C. & Greengard, P. (1986) Proc. Nati. Acad. Sci. USA 83, 4253-4257. 7. Lou, L. L., Lloyd, S. J. & Schulman, H. (1986) Proc. Nati. Acad. Sci. USA 83, 9497-9501. 8. Miller, S. G. & Kennedy, M. B. (1986) Cell 44, 861-870. 9. Schworer, C. M., Colbran, R. J. & Soderling, T. R. (1986) J. Biol. Chem. 261, 8581-8584. 10. Lai, Y., Nairn, A. C., Gorelick, F. & Greengard, P. (1987) Proc. NatI. Acad. Sci. USA 84, 5710-5714. 11. Lickteig, R., Shenolikar, S., Denner, L. & Kelly, P. T. (1988) J. Biol. Chem. 263, 19232-19239. 12. Lisman, J. E. & Goldring, M. A. (1988) Proc. Nati. Acad. Sci. USA 85, 5320-5324. 13. Hardie, G. (1988) Nature (London) 335, 592-593. 14. Kelly, P. T., Weinberger, R. P. & Waxham, M. N. (1988) Proc. Nati. Acad. Sci. USA 85, 4991-4995. 15. House, C. & Kemp, B. E. (1987) Science 238, 1726-1728.
Proc. Natl. Acad. Sci. USA 87 (1990)
1277
16. Kemp, B. E., Pearson, R. B., Guerriero, V., Jr., Bagchi, 1. C. & Means, A. R. (1987) J. Biol. Chem. 262, 2542-2548. 17. Kennelly, P. J., Edelman, A. M., Blumenthal, D. K. & Krebs, E. G. (1987) J. Biol. Chem. 262, 11958-11%3. 18. Payne, M. E., Fong, Y.-L., Ono, T., Colbran, R. J., Kemp, B. E., Soderling, T. R. & Means, A. R. (1988) J. Biol. Chem. 263, 7190-7195. 19. Colbran, R. J., Fong, Y.-L., Schworer, C. M. & Soderling, T. R. (1988) J. Biol. Chem. 263, 18145-18151. 20. Thiel, G., Czernik, A. J., Gorelick, F., Nairn, A. C. & Greengard, P. (1988) Proc. Nati. Acad. Sci. USA 85, 6337-6341. 21. Schworer, C. M., Colbran, R. J., Keefer, J. R. & Soderling, T. R. (1988) J. Biol. Chem. 263, 13486-13489. 22. Miller, S. G., Patton, B. L. & Kennedy, M. B. (1988) Neuron 1, 593-604. 23. Bennett, M. K. & Kennedy, M. B. (1987) Proc. Nati. Acad. Sci. USA 84, 1794-1798. 24. Hanley, R. M., Means, A. R., Ono, T., Kemp, B. E., Burgin, K. E., Waxham, N. & Kelly, P. T. (1987) Science 237, 293297. 25. Lin, C. R., Kapiloff, M. S., Durgerian, S., Tatemoto, K., Russo, A., Hanson, P., Schulman, H. & Rosenfeld, M. G. (1987) Proc. Nati. Acad. Sci. USA 84, 5962-5966. 26. Waxham, M. N., Aronowski, J. & Kelly, P. T. (1989) J. Biol. Chem. 264, 7477-7482. 27. Kelly, P. T. & Shenolikar, S. (1987) Methods Enzymol. 139, 690-714. 28. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987) Methods Enzymol. 154, 367-382. 29. Pearson, R. B., Woodgett, J. R., Cohen, P. & Kemp, B. E. (1985) J. Biol. Chem. 260, 14471-14476. 30. Hanson, P. I., Kapiloff, M. S., Lou, L. L., Rosenfeld, M. G. & Schulman, H. (1989) Neuron 3, 59-70.
Mutagenesis of Thr-286 in monomeric Ca2+/calmodulin-dependent protein kinase II eliminates Ca2+/calmodulin-independent activity (bacterial expression/autophosphorylation/pseudosubstrate)
M. NEAL WAXHAM*, JAROSLAW ARONOWSKI*, SARA A. WESTGATE*, AND PAUL T. KELLYt Departments of *Neurology and tNeurobiology and Anatomy, The University of Texas Medical School at Houston, P.O. Box 20708, Houston TX 77225
Communicated by Philip Siekevitz, November 13, 1989
ABSTRACT
We have examined the role of Thr-286 auto-
phosphorylation in the autoregulation of Ca2+/calmodulindependent protein kinase U. Using site-directed mutagenesis, we have substituted alanine or serine for Thr-286, or isoleucine for Arg-283, in the 50-kDa subunit of the kinase and expressed each protein in bacteria. Activation and autophosphorylation of all four enzymes were stringently dependent on Ca2+/ calmodulin, indicating that neither Arg-283 nor Thr-286 is an absolute requirement for the pseudosubstrate inhibition of the enzyme. Autophosphorylation of the Ile-283 or Ala-2%6 enzyme generated little, if any, Ca2+/calmodulin-independent kinase activity, unlike the parent (Thr-286) or Ser-286 enzyme. The enzymes expressed in bacteria are predominantly monomeric, indicating that the generation of Ca2+/calmodulin-independent activity does not require the cooperative interactions of subunits normally present in the brain holoenzyme.
Ca2+/calmodulin-dependent protein kinase II (CaM-KII) is the predominant protein kinase in mammalian brain and is a major protein component of neuronal postsynaptic densities (1, 2). When activated, CaM-KII phosphorylates a variety of substrates, thereby potentially modulating enzymes and proteins important in neuronal function and plasticity. A dynamic feature of CaM-KII is its autoregulatory property in which Ca2+/CaM-dependent autophosphorylation converts the kinase into a catalytic entity that no longer requires Ca2+/CaM for activity (3-11). This self-regulatory property, together with the subcellular localization of CaM-KII at postsynaptic densities, has led to hypotheses that implicate CaM-KII as a key element in mechanisms encoding the history of synaptic activity (12), a primary event in learning and memory. CaM-KII is a member of a growing family of enzymes autoregulated by pseudosubstrate inhibitory sequences (13). Pseudosubstrates possess substrate-like properties that enable them to bind at or near an enzyme's catalytic and/or substrate-binding domain, thereby inhibiting activity (14-19). The regulation of CaM-KII has been documented through the use of synthetic peptide inhibitors that contain the CaMbinding domain of the enzyme (14-19). Kelly et al. (14) have shown that the pseudosubstrate domain includes an autophosphorylation site (Arg-Gln-Glu-Thr, residues 283-286) on the N-terminal side of the CaM-binding domain. The recent sequencing of autophosphorylated peptides has suggested that Thr-286 in the pseudosubstrate domain is autophosphorylated during generation of Ca2+/CaM-independent kinase activity (20-22). The cloning (23-25) and expression of CaM-KII in bacteria (26) have allowed us to examine the effects of amino acid mutations at Arg-283 and Thr-286 on the generation of the Ca2+/CaM-independent form of CaM-KIT. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Coincidentally, we have also addressed the role of Arg-283 and Thr-286 in the pseudosubstrate regulation of CaM-KI.
MATERIALS AND METHODS Mutagenesis and Isolation of Bacterially Expressed CaMKU. We previously reported (26) the expression in bacteria of the 50-kDa subunit of CaM-KII using the expression vector pK223-2. The original construct contained a cDNA encoding three additional amino acids at the N terminus of the expressed protein. We have reconstructed the 50-kDa cDNA so that the expressed protein now starts at the initiating methionine residue of native CaM-K11. This modification increased the amount of expressed 50-kDa subunit =5-fold. Growth of bacteria and induction of CaM-K11 protein have been described (26). Hypotonic lysates of bacterial protoplasts were clarified by centrifugation at 50,000 X g for 20 min at 40C to obtain soluble and particulate fractions. Soluble and particulate fractions were assayed for Ca2"/ CaM-dependent kinase activity using a synthetic peptide as substrate (see below); 90% Ca2+/
A) Ala-286 10 Co 0
T-
V
SUBSTRATE (NTP, uM)
1/S
C) Ala-286
Inm
lV
D) Thr-286
30
3
25
2,
!5
K m 1.8 uM Vma = 0.20 uMoV
20
21!0
min/mg
-15
-1 5
-
0)
V
V
2
10
5 -0.2 -0.1
0.0
0.1
0.2
1/S
0.3
0.4
0.5
01 -0.2 -0.1
Km7.1 uM Vmax = 0.45 umoVmirnmg 0.0
0.1
0.2
0.3
0.4
0.5
1/S
FIG. 2. Kinetic analysis of bacterially expressed 50-kDa subunits at various concentrations of synthetic peptide (NTP). Phosphorylation reactions were carried out with the Ca2+/CaM-dependent (e) or Ca2+/CaM-independent (0) forms of the different enzymes; reactions were performed in duplicate and each mixture contained 50 ng of 50-kDa subunits. (A) Peptide phosphorylation by the Ala-286-substituted enzyme. (B-D) Double-reciprocal plots of peptide phosphorylation with Ser-286, Ala-286, or Thr-286 enzyme, respectively. The apparent Km and Vmax for each enzyme are included in the appropriate panel.
1276
Biochemistry: Waxham et al. 100. 80
A
Blue-Dex.
-
0,0 Arg-283 AA Ile-283
A.
-
c
8 40
A
,
/
60
Proc. Natl. Acad. Sci. USA 87 (1990)
CO
x
A Holoenzyme
* Thr-286 o Ala-286
'T5 0.2
C,,
20
Cyto-C
+ +
0.6 0.4
-.
I//-
-
I');o
BSA
-
0.0 ~ ~ ~ ~ ~
~
~
~
~
~
~
~
~~~~~-
10
AI.
0
100. 0
80 .
0.
60 .
60
30
90
120
150
*
Arg-283
A' Ile-283
40 .
c
(D Q
20 . 0. 20
.
40
140
60
80 100 Time (sec)
120
30
35
FIG. 4. Gel filtration analysis of rat forebrain CaM-KII and bacterially expressed CaM-KlI. Each enzyme was purified by phosphocellulose chromatography and analyzed by gel filtration. Fractions (1 ml) were collected and 20 ,ul was analyzed for Ca2+/ CaM-dependent protein kinase activity; A, rat brain holoenzyme; *, bacterially expressed Thr-286 enzyme; o, Ala-286 enzyme. Elution peaks for the molecular mass standards Blue dextran 2000 (2000 kDa), bovine serum albumin (BSA, 67 kDa) and cytochrome c (12.3 kDa) are indicated.
..
. _.
25 20 Volume (mL)
210
B
0X c
180
15
140
FIG. 3. Kinetic comparisons of bacterially expressed Arg-283 (circles) and Ile-283 (triangles) enzymes. (A) Equivalent amounts of each 50-kDa subunit (100 ng) were examined for Ca2+/CaMdependent phosphorylation of 30 ,uM syntide (cpm x 10-3, open symbols) or 50-kDa autophosphorylation (cpm x 10-2, filled symbols); reactions varied from 20 to 180 sec. (B) The generation of Ca2+/CaM-independent activity was analyzed for equivalent amounts of Arg-283 or Ile-283 enzyme; each enzyme was autophosphorylated for 30, 60, or 120 sec and then allowed to phosphorylate substrate in the presence of Ca2+ or EGTA. Independent activity (%) was calculated from ratios of substrate phosphorylation in EGTA vs. c2+.
Ca2~
Arg-283 contributes to the efficient generation of Ca2+/ CaM-independent activity. Gel filtration analyses (Fig. 4) showed that both the Thr286 and Ala-286 enzymes were eluted between bovine serum albumin (67 kDa) and cytochrome c (12.3 kDa), indicating that the enzymes expressed in bacteria are predominantly monomeric. Small amounts of the Thr-286 enzyme were eluted ahead ofbovine serum albumin, suggesting that a small proportion of this preparation may contain oligomeric forms of the enzyme. None of these apparent oligomers was present in the Ala-286 preparations. CaM-KII isolated from rat forebrain was eluted just after Blue dextran (2000 kDa) but well before bovine serum albumin, consistent with previous reports that the brain enzyme is multimeric (1, 27). Many kinases are regulated by pseudosubstrate inhibitory domains (13-18). While CaM-KII is among the enzymes activated by Ca2+/CaM, it contains a bona fide phosphorylation sequence within its inhibitory domain. Moreover, although many kinases undergo autophosphorylation, subsequent functional changes are much less dramatic than those displayed by CaM-KII. The pseudosubstrate domain of CaMKII appeared to require the consensus phosphorylation se-
quence Arg-Gln-Glu-Thr286 (14, 19), whose phosphorylation resulted in the generation of Ca2+/CaM-independent activity. By coupling the pseudosubstrate and CaM-binding domains, CaM-KII regulates the efficiency of Thr-286 phosphorylation; in the absence of Ca2+/CaM the former is an inhibitor, while in their presence it becomes a substrate. This hypothesis was initially supported by studies which showed that the intact sequence Arg-Gln-Glu-Thr in longer pseudosubstrate peptides was required for efficient inhibition (IC50 = 2 ,uM) of the Ca2+/CaM-independent form of CaMKII (14, 19). The latter studies also implied that disruption of critical determinants in this sequence, namely Arg-283 and Thr-286, may eliminate pseudosubstrate inhibition. We have shown that arginine at position 283 and threonine at position 286 are not absolute requirements for a functional pseudosubstrate inhibitory domain. The Ile-283, Ala-286, and Ser-286 enzymes each remained completely dependent on Ca2+/CaM for activity and followed similar CaM-activation kinetics when compared to the parent enzyme. Thus, the precise function and location of the pseudosubstrate domain in the native kinase are clearly more complex than studies with synthetic peptides had predicted. Further site-directed mutagenesis studies may reveal the critical amino acids responsible for the pseudosubstrate inhibition in native CaM-KII. CaM-KII purified from rat forebrain is a multisubunit holoenzyme containing two distinct protein subunits (50 and 60 kDa). It is clear that the kinase's ability to convert to a Ca2+/CaM-independent enzyme does not require both subunits (26), and the gel filtration data presented here indicate that the generation of autonomous catalytic activity can reside in a single monomeric subunit. A recent report (30) describing the transient expression of the 50-kDa subunit in eukaryotic cells and subsequent analysis of whole-cell extracts also determined that substitution of alanine for Thr-286 produced an enzyme that displayed very little Ca2+/CaMindependent activity after prior autophosphorylation. Interestingly, the enzyme expressed in eukaryotic cells is a holoenzyme of 6-10 subunits. We have reached similar conclusions with bacterially expressed enzymes and have shown that monomers of the 50-kDa subunit exhibit Ca2+/ CaM-independent activity after autophosphorylation. This indicates that cooperativity between subunits is not required for generation of Ca2+/CaM-independent activity.
Biochemistry: Waxham et al. We thank Allison Stock for technical assistance, Vikram Dabhi for help with the gel filtration studies, and Randall Kincaid for CaMSepharose. This work was supported by U.S. Public Health Service Grant NS26086. 1. Kennedy, M. B., Bennett, M. K. & Erondu, E. (1983) Proc. Natl. Acad. Sci. USA 80, 7357-7361. 2. Kelly, P. T., McGuinness, T. & Greengard, P. (1984) Proc. NatI. Acad. Sci. USA 81, 945-949. 3. Shields, S. M., Vernon, P. J. & Kelly, P. T. (1984) J. Neurochem. 43, 1599-1609. 4. Kelly, P. T., Yip, R., Shields, S. M. & Hay, M. (1985) J. Neurochem. 45, 1620-1634. 5. Saitoh, T. & Schwarz, J. H. (1985) J. Cell Biol. 100, 835-842. 6. Lai, Y., Nairn, A. C. & Greengard, P. (1986) Proc. Nati. Acad. Sci. USA 83, 4253-4257. 7. Lou, L. L., Lloyd, S. J. & Schulman, H. (1986) Proc. Nati. Acad. Sci. USA 83, 9497-9501. 8. Miller, S. G. & Kennedy, M. B. (1986) Cell 44, 861-870. 9. Schworer, C. M., Colbran, R. J. & Soderling, T. R. (1986) J. Biol. Chem. 261, 8581-8584. 10. Lai, Y., Nairn, A. C., Gorelick, F. & Greengard, P. (1987) Proc. NatI. Acad. Sci. USA 84, 5710-5714. 11. Lickteig, R., Shenolikar, S., Denner, L. & Kelly, P. T. (1988) J. Biol. Chem. 263, 19232-19239. 12. Lisman, J. E. & Goldring, M. A. (1988) Proc. Nati. Acad. Sci. USA 85, 5320-5324. 13. Hardie, G. (1988) Nature (London) 335, 592-593. 14. Kelly, P. T., Weinberger, R. P. & Waxham, M. N. (1988) Proc. Nati. Acad. Sci. USA 85, 4991-4995. 15. House, C. & Kemp, B. E. (1987) Science 238, 1726-1728.
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