(Received for publication, October 21, 1994)
From the
Phosphorylation of rat non-muscle caldesmon by cdc2 kinase causes reduction in most of caldesmon's properties, including caldesmon's binding to actin, myosin, and calmodulin, as well as its inhibition of actomyosin ATPase. We have generated and characterized the COOH terminus of caldesmon mutants lacking mitosis-specific phosphorylation sites, because the COOH-terminal half of caldesmon contains all 7 putative Ser or Thr sites for cdc2 kinase. Codons for the 7 putative Ser or Thr residues have been mutated to Ala, and resultant mutants were bacterially expressed. Analyses of the phosphopeptide maps of these mutants have identified 6 sites, including Ser-249, Ser-462, Thr-468, Ser-491, Ser-497, and Ser-527 as the mitosis-specific phosphorylation sites, whereas the phosphorylation of the remaining site, Thr-377, is not detected by this assay method. Actin binding experiments have suggested that 5 sites including Ser-249, Ser-462, Thr-468, Ser-491, and Ser-497 are important for the phosphorylation-dependent reduction in actin binding. Characterization of a mutant lacking all 7 Ser or Thr sites (7-fold mutant) has revealed that 7-fold mutation eliminates all phosphorylation sites by cdc2 kinase. While the in vitro properties of the 7-fold mutant, including actin, myosin, and calmodulin binding and inhibition of actomyosin ATPase, are very similar to those of nonmutated protein, such properties are not affected by the treatment with cdc2 kinase in contrast to nonmutated protein. This mutant should thus be useful to explore the functions of the mitosis-specific phosphorylation of caldesmon.
Microfilaments in cultured cells show drastic changes in their organization during mitosis(1, 2) . Little is known regarding the molecular bases for these changes. Because a variety of actin-binding proteins regulate actin assembly and organization of microfilaments(3, 4) , it is likely that they are involved in the changes in microfilament organization during mitosis. We have demonstrated previously that non-muscle caldesmon is specifically dissociated from microfilaments during mitosis, apparently as a consequence of the mitosis-specific phosphorylation by cdc2 kinase(5, 6) . Others also have shown that cdc2 kinase phosphorylates smooth muscle caldesmon(7) . We suggested that the dissociation of caldesmon may cause, at least in part, the alterations in the microfilament organization during mitosis, because caldesmon is a regulatory protein implicated in the control of actomyosin interactions and/or microfilament organization.
In a
reconstituted system, caldesmon functions in the following three ways
(see (8, 9, 10) for reviews). First,
caldesmon inhibits the tropomyosin-stimulated, actin-activated ATPase
of
myosin(11, 12, 13, 14, 15, 16) .
This inhibition is attenuated by Ca/calmodulin, which
reverses actin binding by caldesmon. Second, caldesmon, together with
tropomyosin, regulates stability of actin filaments in vitro.
Caldesmon stimulates actin binding of tropomyosin which increases the
stabilization of actin filament structure(17, 18) . In
addition, caldesmon and tropomyosin together also inhibit the actin
severing and capping activities of gelsolin(19, 20) .
Third, caldesmon stimulates actin
polymerization(21, 22) . Thus, the dissociation of
caldesmon from microfilaments may release the inhibition of actomyosin
ATPase, destabilize microfilament structure, and/or change actin
polymerization.
The cdc2 kinase constitutes a subunit of MPF (maturation or M-phase promoting factor), a cell cycle control element that initiates all of the events seen during mitosis in eukaryotes (see (23, 24, 25) for review). The finding that caldesmon is phosphorylated by cdc2 kinase poses a question: how does this cell cycle control element regulate microfilament organization during mitosis? One way to address this question is to generate mutant caldesmons lacking phosphorylation sites and to see what effects mutant caldesmons have on the microfilament organization during mitosis. As a first approach toward this goal we have generated mutant caldesmons lacking phosphorylation sites through site-directed mutagenesis. Rat non-muscle caldesmon contains seven putative phosphorylation sites (Ser or Thr)-Pro, for cdc2 kinase, all of which are mutated to Ala. Analyses of phosphorylation patterns of the mutants have allowed us to identify the phosphorylation sites of rat non-muscle caldesmon by cdc2 kinase. We have generated and characterized a mutant, in which all seven sites are mutated. We have found that such 7-fold mutation completely eliminates phosphorylation by cdc2 kinase and that treatment with cdc2 kinase does not affect the in vitro properties of the mutant, including actin, myosin, and calmodulin binding and inhibition of actomyosin ATPase.
We grew BL21(DE3)plysS in M9ZB medium containing 50
µg/ml of ampicillin and 25 µg/ml of chloramphenicol. The
expression of the target protein was induced by the addition of 0.4
mM of isopropyl-1-thio-galactopyranoside when culture reached
an OD of 0.5-0.6. After 2 h of induction, the
bacteria were lysed by sonication in 0.1 M NaCl and 20 mM Tris-HCl of pH 8.0, and centrifuged at 10,000 rpm for 10 min. The
supernatant was heat-treated at 95 °C for 10 min, cooled on ice for
30 min, and then centrifuged again. The supernatant was then applied on
a calmodulin-Sepharose column (Pharmacia Biotech Inc.). After extensive
washing of the column with 50 mM imidazole buffer of pH 7.0
containing 150 mM NaCl, 0.5 mM CaCl
, 0.2
mM dithiothreitol, and 0.2 mM phenylmethylsulfonyl
fluoride, D3 protein was purified by the elution with the same buffer
except that 0.5 mM CaCl
was replaced with 2 mM EGTA. Approximately 2 mg of D3 protein were prepared from 10
liters of bacterial culture.
Actin Binding Assay-Actin binding of D3 peptide and its mutants was performed in essentially the same way as described for actin binding of intact caldesmon(26) . The assay conditions were 20 mM imidazole buffer of pH 7.0, 100 mM KCl, 12 µM F-actin and varying amounts of D3 protein and its mutants from 0 to 8 µM. The reaction mixtures were centrifuged for 20 min at 26 p.s.i. in a Beckman Airfuge. Both supernatants and pellets dissolved in the same volume of SDS sample buffer were analyzed by SDS-polyacrylamide gel electrophoresis, and the amounts of D3 protein were determined by densitometry as described (26) .
Phosphorylation of D3 protein and its mutants by cdc2 kinase was performed as described(26) . Phosphorylated proteins were purified by heat treatment and examined for actin binding as described(26) .
Figure 1: Mapping of rat non-muscle caldesmon cDNAs.
The largest open reading frame starts with Met at position 724, which does not begin with a typical Kozak consensus sequence. The reading frame terminates at position 2316 and encodes 531 amino acids. Peptide sequences of two CNBr fragments of 83-kDa non-muscle caldesmon match the amino acid sequences derived from the DNA sequence, confirming the authenticity of the cDNAs(6) . The deduced protein molecular weight is 60,607 which is significantly less than the molecular weight (83,000) estimated by SDS-gel electrophoresis. Chick and human caldesmons have also been reported to show a higher molecular weight on SDS gels(29, 30) .
Figure 2: Amino acid sequence of rat non-muscle caldesmon. Putative phosphorylation sites by cdc2 kinase are double underlined. An arrowhead indicates the start of D3 protein.
Figure 3: Bacterial expression of D3 protein (the COOH terminus of rat non-muscle caldesmon, Glu-235 to Val-531). D3 protein is native in terms of its solubility and calmodulin binding ability. SDS gel analyses of a heat-stable fraction of total soluble proteins (lane 1) and purified D3 protein by calmodulin affinity column chromatography (lane 2). Lane M, molecular mass markers (200, 117, 94, 68, 42, 30, and 21 kDa).
Figure 8:
Effects of mitosis-specific
phosphorylation on actin binding of phosphorylation mutants. Wild type
D3 protein as well as two kinds of double mutant, 4-, 5-, 6-, and
7-fold mutants were phosphorylated by cdc2 kinase and assayed for actin
binding (shown with closed symbols). As a control, actin
binding was also measured without phosphorylation (shown with open
symbols). A, actin binding of D3 protein (,
)
and two kinds of double mutants (Ser-462/Thr-468 (
,
) or
Ser-491/Ser-497 (
,
); B, actin binding of D3
(
,
), 4-fold (Ser-462/Tre-468/Ser-491/Ser-497 ((
,
)), 5-fold (Ser-462/Tre-468/Ser-491/Ser-497/Ser-527 ((
,
)), and 6-fold (Thr-377/Ser-462/Thr-468/Ser- 491/Ser-497/Ser-527
(
,
); C, actin binding of D3 protein ((
,
)) and 7-fold
(Ser-249/Thr-377/Ser-462/Thr-468/Ser-491/Ser-497/Ser-527 ((
,
)
mutants.
Analyses of phosphorylation sites using two-dimensional phosphopeptide mapping have revealed that cdc2 kinase phosphorylates D3 protein at the same sites as it does intact caldesmon. A two-dimensional phosphopeptide map (Fig. 4A) of D3 protein phosphorylated by cdc2 kinase is identical to that produced with authentic rat non-muscle caldesmon (compare with Fig. 2B of (26) ). These results further support the nativeness of D3 protein and indicate that D3 protein can be used for identification of all cdc2 phosphorylation sites through in vitro mutagenesis.
Figure 4: Two-dimensional maps of wild D3 and mutant D3 proteins lacking the phosphorylation sites at Ser-249, Thr-377, and Ser-527. A single mutation at Ser-249 (B), at Thr-377 (C), or at Ser-527 (D) to Ala was made by oligonucleotide-directed mutagenesis. The mutant proteins were expressed, phosphorylated by cdc2 kinase and compared with the two-dimensional pattern produced from wild type D3 protein (A). The origin is marked by an ``o''. The changes in phosphopeptide spots are indicated by arrowheads with letters.
First, the effects of three single mutations of Ser-249 to Ala, Thr-377 to Ala and Ser-527 to Ala were examined (Fig. 4). The Ser-249 to Ala mutation (B) causes disappearance of two major (spots a and b) and one minor spots (indicated by c), suggesting that Ser-249 is a phosphorylation site by cdc2 kinase. This assignment is consistent with the phosphoamino acid analyses showing that spots a, b, and c are phosphoserine (data not shown). Likewise, the single point mutation at Ser-527 (D) reproducibly eliminates one major spot (spot d). This is again consistent with the phosphoamino acid analyses which reveal that spot d contains phosphoserine. Ser-527 is thus identified as a site of phosphorylation. On the other hand, Thr-337 is not detected as a major phosphorylation site by this assay method. A phosphopeptide map (C) generated by the Thr-377 to Ala mutant appears very similar to that (A) produced by wild D3 protein.
We have next examined whether Ser-462 and Thr-468 are phosphorylation sites (Fig. 5). A single point mutation (B) at Ser-462 to Ala causes disappearance of two spots, e and f. On the other hand, a single point mutation at Thr-468 to Ala (C) eliminates two spots, g and f. Phosphoamino acid analyses have revealed that spot e is phosphoserine, whereas spot g is phosphothreonine (phosphoamino acid of spot f is not determined). These results suggest that (a) spot e is Ser-462; (b) spot g is Thr-468; and (c) spot f is generated by double phosphorylation at both Ser-462 and Thr-468. A double mutation at both sites (D) eliminates one additional spot h in addition to the above three spots, e-g. Phosphoamino acid analyses have revealed that spot h contains both phosphoserine and phosphothreonine. These results may be explained in the following way: spot h may be a mixture of two phosphopeptides which are generated by single phosphorylation at either Ser-462 or Thr-468 and co-migrate at the same position of h; thus only double mutations but not single mutations can eliminate the spot h.
Figure 5: Two-dimensional maps of mutants lacking the phosphorylation sites at Ser-462 and/or Thr-468. A, map of wild D3; B, a single mutation at Ser-462 to Ala; C, a single mutation at Thr-468 to Ala; D, a double mutation at both Ser-462 and Thr-468 to Ala.
The identification of spot h as the phosphorylation site at Ser-462 or Thr-468 is further supported by a phosphopeptide map generated by phosphorylation of a synthetic peptide (KGSVFSSPSASGTPNKE) corresponding to these sites. The peptide was phosphorylated in vitro by cdc2 kinase, digested with trypsin, and analyzed by two-dimensional mapping. The analysis of a mixture of phosphorylated caldesmon and synthetic peptide has revealed that a spot generated by the peptide co-migrates with the spot h (data not shown).
Ser-491 and Ser-497 also are phosphorylation sites (Fig. 6). A single mutation at Ser-497 (C) eliminates a cluster of spots (indicated by i) and generates a new spot j, indicating that the cluster i is generated by phosphorylation at Ser-497. A single mutation at Ser-491 (B), on the other hand, does not seem to change the peptide pattern significantly. However, a double mutation at both sites (D) eliminates not only the cluster i, but also a spot, k. In addition, the new spot, j, disappears, suggesting that the new spot, j, is a product of Ser-491 phosphorylation. These results indicate that both Ser-491 and Ser-497 are involved in the phosphorylation and suggest that spot k may contain two phosphopeptides, each of which is singly phosphorylated at either Ser-491 or Ser-497, but co-migrates at the same position, k.
Figure 6: Two-dimensional maps of mutants lacking the phosphorylation sites at Ser-491 and Ser-497. A, map of wild D3; B, a single mutation at Ser-491 to Ala; C, a single mutation at Ser-497 to Ala; D, a double mutation at both Ser-491 and Ser-497 to Ala.
Consistent with the above explanation, spot k is shown to contain phosphoserine, although phosphoamino acid analyses failed to reveal the content of the spots in the cluster i. It is also noticed that spot g, which is assigned as Thr-468, disappears in the maps of the double mutation. Although the reason for this is unknown, one possible explanation is that the phosphorylation at Thr-468 may occur only after the phosphorylation at the Ser-491 and Ser-497 sites. This explanation is consistent with the disappearance of spot f, which is thought to be produced by the double phosphorylation at Ser-462 and Thr-468.
We have further examined
phosphopeptide maps of 6-fold (mutations at Thr-377, Ser-462, Thr-468,
Ser-491, Ser-497, and Ser-527 to Ala) and 7-fold (mutations at Ser-249,
Thr-377, Ser-462, Thr-468, Ser-491, Ser-497, and Ser-527 to Ala)
mutants. As Fig. 7B shows, the map of the 6-fold mutant
yields two major (a and b) and three minor (c, l, and m) spots, of which three spots, a-c, are assigned as Ser-249 (see Fig. 4B). Consistent with this assignment, these three
spots, a-c, disappear in the map of 7-fold mutants (Fig. 7B). In addition, two other minor spots, l and m, also disappear, although we do not know why the
two minor spots disappear by 7-fold mutation. Thus the map of 7-fold
mutants shows virtually no phosphopeptide spots, indicating that all
cdc2 phosphorylation sites are blocked by 7-fold mutation. This is
consistent with the observation that the 7-fold mutant does not
incorporate any significant P radioactivity in
phosphorylation experiments with cdc2 kinase (data not shown).
Figure 7: Two-dimensional maps of mutants lacking the six and seven phosphorylation sites. A, map of wild D3; B, 6-fold mutation (Thr-377/Ser-462/Thr-468/Ser-491/Ser-497/Ser-527 to Ala); C, 7-fold mutation (Ser-249/Thr-377/Ser-462/Thr-468/Ser-491/Ser-497/Ser-527 to Ala). Note that 7-fold mutation almost completely eliminates phosphopeptides. In A, the amino acid residue number of the phosphorylation sites are indicated by the numbers in parentheses.
First, actin binding of
unphosphorylated proteins was measured as controls. As Fig. 8shows, all unphosphorylated mutants exhibit actin binding
profiles almost identical to that of unphosphorylated wild type D3
protein. These results suggest that the mutations at these sites do not
affect actin binding properties to any significant extent.
Phosphorylation of wild type D3 protein with cdc2 kinase causes almost
complete loss of its actin binding ability, where the apparent binding
constant is reduced from 5 10
to less than 10
M
. This extent of reduction in
binding constants is similar to that observed with intact
caldesmon(26) .
Blockade of the phosphorylation sites
abolishes phosphorylation-dependent reduction in actin binding. As Fig. 8, A and B, show, the double and 4-fold
mutations partially inhibit this phosphorylation-dependent reduction in
actin binding. The apparent binding constants of the phosphorylated
double and 4-fold mutant D3 proteins are increased to 5
10
and 2
10
M
, respectively. Further mutation at
Ser-527 (5-fold mutation) or at both Ser-527 and Thr-377 (6-fold
mutation) shows only a slight increase on actin binding upon
phosphorylation over the actin binding of 4-fold mutant (B).
The seventh mutation at Ser-249, however, yields a large effect on the
phosphorylation-dependent actin binding. Actin binding of 7-fold mutant
is not decreased after phosphorylation by cdc2 kinase (C). The
absence of the phosphorylation effect is consistent with the
observation that cdc2 kinase is unable to phosphorylate the 7-fold
mutant.
7-Fold mutation also attenuates the effects of phosphorylation on caldesmon's ability to inhibit actomyosin ATPase. As Fig. 9shows, phosphorylation by cdc2 kinase completely abolishes the inhibition of actomyosin ATPase by D3 protein. On the contrary, the ability of the 7-fold mutant to inhibit ATPase activity is little affected by treatment with cdc2 kinase.
Figure 9:
Effects of phosphorylation on inhibition
of actomyosin ATPase by D3 protein and 7-fold mutant. Relative ATPase
activities were plotted against concentration of D3 protein (,
) or 7-fold mutant (
,
). Note that phosphorylated
D3 protein shows no inhibition of ATPase, whereas the inhibition of
7-fold mutant is not affected by the treatment with cdc2 kinase. Open symbols, unphosphorylated; closed symbols,
treated with cdc2 kinase.
We have also examined the effects of 7-fold mutation on caldesmon's other properties including myosin binding, stimulation of actin-tropomyosin binding, and calmodulin regulation of actin binding. 7-fold mutation does not affect these properties (data not shown). Furthermore, as expected by the lack of phosphorylation in 7-fold mutant, cdc2 kinase does not alter these properties of 7-fold mutant (data not shown).
In this paper, we have generated and characterized a variety of mutants of the COOH terminus of rat non-muscle caldesmon lacking cdc2 phosphorylation sites. Our studies have revealed the following important points; (a) among the seven putative cdc2 sites of rat non-muscle caldesmon, six sites (Ser-249, Ser-462, Thr-468, Ser-491, Ser-497, and Ser-527) are identified as the major cdc2 phosphorylation sites, whereas the remaining site of Thr-377 is not detected as a major phosphorylation site; (b) five sites (Ser-249, Ser-462, Thr-468, Ser-491, and Ser-497) are more important than the other two sites (Ser-527 and Thr-377) in controlling caldesmon-actin interactions; (c) the 7-fold mutation eliminates all cdc2 phosphorylation sites of rat non-muscle caldesmon; and (d) in vitro properties of the 7-fold mutant, including actin binding and inhibition of actomyosin ATPase, are affected neither by the mutation itself nor by phosphorylation with cdc2 kinase.
Our work suggests that five sites including Ser-249, Ser-462, Thr-468, Ser-491, and Ser-497 are equally important. Among the six phosphorylation sites, four sites, including Ser-462, Thr-468, Ser-491, and Ser-497, are well conserved among rat, human, and chick caldesmons (6, 31) . In addition, Mak et al. (32) reported that cdc2 mainly phosphorylates these four sites of chick smooth muscle caldesmon. However, the mutation at these four sites does not completely block the phosphorylation-dependent inhibition of actin binding of rat non-muscle caldesmon. Phosphorylation at Ser-249 is equally important for reducing actin binding of rat non-muscle caldesmon (Fig. 8), although phosphorylation at the site corresponding to Ser-249 was not reported with chick smooth muscle caldesmon. In contrast, the mutations at Thr-377 and Ser-527 show meager effects on the phosphorylation-dependent actin-caldesmon interactions. Both 5- and 6-fold mutants shows only a slight increase in actin binding over the 4-fold mutant upon phosphorylation.
Fig. 10shows the domain structure of rat non-muscle caldesmon with the sites of phosphorylation indicated. It should be noted that all these phosphorylation sites except for Ser-527 are localized outside of actin and calmodulin-binding domains. This is consistent with our finding that all mutants lacking phosphorylation sites shows normal actin and calmodulin binding. This notion suggests that the domain(s) containing these phosphorylation sites would interact with the strong actin binding domain (Leu-392 to Ile-424). Alternatively, the phosphorylation at these sites would induce conformational changes, which result in low actin and calmodulin binding of caldesmon. These possibilities are intriguing, since one of the sites, Ser-249, is, in the primary sequence, located far from the strong actin binding domain.
Figure 10: Domain structure of rat non-muscle caldesmon. The domain structure suggested for chick smooth muscle caldesmon by others (see (34) for example) is adopted to assign myosin-, actin-, and calmodulin-binding domains of rat non-muscle caldesmon according to the sequence homology between chick and rat caldesmons. Numbers on the top of the figure show the putative phosphorylation sites containing either SP or TP. Of these, Ser-249, Ser-462, Thr-468, Ser-491, Ser-497, and Ser-527 are identified as the phosphorylation sites by cdc2 kinase in this study.
We have used site-directed mutagenesis to identify cdc2 phosphorylation sites. The mutagenesis method has an advantage over the conventional method of peptide sequencing of purified phosphopeptides. It allows us to prepare and characterize mutants at the same time. However, the mutagenesis method is an indirect way to identify sites, and it is possible that the mutagenesis method may not be as sensitive as the conventional method.
Indeed, this may explain one of the differences between our work and that of Mak et al.(32) . Although we have not detected the phosphorylation of Thr-377, Mak et al. (32) identified Ser-582 (corresponding to rat Thr-377) of chick smooth muscle caldesmon through peptide sequencing of purified phosphopeptides(32) . Because the level of the phosphorylation at Ser-582 was reported to be very low (about 13% of total phosphorylation), the mutagenesis method may not detect phosphorylation at Thr-377. In any case, the phosphorylation at rat Thr-377, if it occurs, appears to be minor. This notion is consistent with the observation that the mutation at this site does not largely affect the phosphorylation-dependent reduction of actin binding; actin binding of the 6-fold mutant is indistinguishable from the 5-fold mutant (Fig. 8).
Our work has identified two other cdc2 phosphorylation sites, Ser-249 and Ser-527, with rat non-muscle cadesmon, which have not been reported with chick smooth muscle caldesmon by Mak et al.(32) . While Ser-527 is unique to mammalian caldesmons(6, 31) , chick smooth muscle caldesmon has Thr-470 whose relative position is similar, although not identical, to the position of Ser-249 of rat caldesmon. This difference can be explained by the fact that the sequences around these two sites are rather different. Alternatively, the central domain of smooth muscle caldesmon may inhibit the phosphorylation of Thr-470.
Redwood et al.(33) have shown that the mutation at Ser-702 (corresponding to Ser-497 of rat non-muscle caldesmon) to Asp reduces the actin binding ability of the mutant, whereas the mutation at Thr-673 (corresponding to Thr-468 of rat non-muscle caldesmon) to Asp does not. Our results, on the other hand, have shown that caldesmons with double mutations at either Ser-462/Thr-468 or Ser-491/Ser-497 show indistinguishable effects on actin binding after phosphorylation by cdc2 kinase (Fig. 8). Together, these results would then suggest that phosphorylation at Ser-462 is more critical for the regulation of actin binding than phosphorylation at Thr-468.
It is possible that the 7-fold mutant behaves as a dominant negative mutant. The 7-fold mutation should eliminate all cdc2 phosphorylation sites of the entire caldesmon molecule, because the phosphopeptide map of the C terminus of caldesmon is exactly the same as that of intact caldesmon. The 7-fold mutation itself does not alter the properties of caldesmon, including binding to actin, calmodulin, and myosin, inhibition of actomyosin ATPase, stimulation of actin-tropomyosin binding, and calmodulin regulation of actin binding. More importantly, cdc2 kinase does not affect these properties of the 7-fold mutant, as expected from the lack of phosphorylation. Thus the mutant, when expressed in cultured cells, would stay bound to microfilaments, whereas endogenous wild type caldesmon is dissociated from microfilaments. This may cause stabilization of microfilaments and/or inhibition of actomyosin ATPase during mitosis. Our next goal is to see what effects the mutant caldesmon has on microfilament organization during mitosis. We are in the process of transfection experiments with wild type and mutant caldesmon in cultured cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18419[GenBank].