Gwl phosphorylation and activation.
Gwl activation during M phase involves its hyperphosphorylation, affecting Gwl's electrophoretic mobility ( and ) and leading to the labeling of more than 20 tryptic phosphopeptides (data not shown). One or more phosphosites in M phase Gwl constitute epitopes for the MPM-2 monoclonal antibody (A). This antibody reacts with many mitotic phosphoproteins, often at motifs targeted by cyclin-dependent kinases (CDKs) that contain phosphoserine or phosphothreonine followed by proline (42
Fig 2 Gwl phosphorylation. (A) M phase but not interphase Gwl carries an MPM-2 epitope. CSF extracts were directly analyzed (M) or treated with calcium (interphase extracts; I). Input lanes contain 10% of the extract subjected to Gwl immunoprecipitation (IP). (more ...)
We have reconstituted a significant fraction of the Gwl activation process in vitro
by treating interphase Gwl (made from baculovirus constructs in Sf9 cultured cells) with CDKs () (also see references 40
). Consistent with the general lack of CDK specificity in vitro
), Cdk1-cyclin B (MPF), Cdk2-cyclin E, and Cdk2-cyclin A are equally efficient in activating Gwl (A and data not shown). CDK-treated interphase Gwl attains at most only ~25% of the kinase activity of M phase Gwl (made in Sf9 cells arrested with the phosphatase inhibitor okadaic acid [OA]; see references 45
). However, following the prescient suggestion of a reviewer, we added the CDK-associated protein Cks2 (Suc1/p9) to the in vitro
activation reaction mixture. This factor was previously reported to promote CDK phosphorylation of other G2
/M regulators, such as Cdc25 (29
). The addition of Cks2 enhanced CDK activation of Gwl by ~4 times, producing an enzyme with activity more comparable to that of Gwl made in the presence of OA (C and D). Gwl activation may therefore not require upstream kinases other than CDKs. Indeed, in our hands, the M phase kinases Plx1, Aurora A, and Nek2 do not contribute to Gwl activation alone or in synergism with CDKs (B); the same is true of PKA and p42 mitogen-activated protein kinase (MAPK) (data not shown).
Gwl undergoes autophosphorylation; that is, active preparations of wild-type Gwl (either endogenous protein purified from M phase extracts or recombinant enzyme made in OA-treated Sf9 cells) become labeled by radioactive ATP. This labeling represents autophosphorylation rather than the action of a contaminating kinase because kinase dead (KD) Gwl does not incorporate 32
) (A and see A) and because a linear correlation exists between the phosphorylation of mutant forms of Gwl and their activities on exogenous substrates (see below). At least 15 tryptic peptides become labeled by Gwl autophosphorylation (data not shown); as explained below, most of these sites are not critical to Gwl activation, but at least one of the sites does play a key role.
Fig 4 Gwl autophosphorylation is primarily intramolecular. (A) Amounts of 25 ng of WT or KD Gwl (both containing a Z tag and made in OA-treated Sf9 cells) were incubated in kinase assays with 250 ng of either heat-inactivated Gwl(Δ300-650) or heat-inactivated (more ...)
To determine whether Gwl autophosphorylation is intra- or intermolecular, we performed two experiments in which a small amount of active, WT Gwl was added to an excess of inactive Gwl (). 32P was incorporated almost exclusively into active Gwl, showing that the autophosphorylation is primarily intramolecular. 32P labeling of active Gwl displays first-order kinetics typical of unimolecular reactions (C and D), supporting the intramolecular autophosphorylation model.
Specific sequences within the long intervening region are not essential for Gwl function.
Gwl's highly conserved kinase domain is split by a poorly conserved ~500-amino-acid insertion (residues 223 to 717 in Xenopus Gwl) ( and ). To establish whether this nonconserved middle region (NCMR) is critical for Gwl activity, we made truncated proteins, including Gwl(Δ241-549), Gwl(Δ400-699), and Gwl(Δ300-650), that collectively remove almost all of the NCMR (A and data not shown). The truncated proteins, as well as WT and KD Gwl, were expressed in Sf9 cells that were either arrested in M phase by OA or untreated and thus mostly in interphase.
Fig 5 Schematic view of Gwl structure and mutant analysis. In WT Xenopus Gwl, the two halves of the conserved kinase domain are orange, the NCMR is yellow, and blue shows regions that distinguish Gwl from other AGC kinases. In the top row, red tick marks indicate (more ...)
Fig 6 Gwl phosphorylation sites and mutagenesis. Xenopus Gwl aligned with human, mouse, and Drosophila counterparts. In vivo mitotic Xenopus Gwl was purified by immunoprecipitation from CSF extracts or by expression in OA-treated Sf9 insect cells with nearly (more ...)
Fig 7 NCMR deletions. (A) WT, KD, and deletion mutant Gwl were purified from Sf9 cells in the presence or absence of OA. Proteins were stained with Coomassie blue; those purified from OA-treated cells were treated with λ phosphate (PPase) before electrophoresis (more ...)
The purified proteins were adjusted to the same molar concentration and assayed for kinase activity (B). The results in C show the assay's dynamic range. The model substrates myelin basic protein (MBP) and endosulfine yielded similar results. The interphase enzymes displayed little or no kinase activity. Surprisingly, when expressed in OA-treated cells, Gwl(Δ241-549), Gwl(Δ400-699), and Gwl(Δ300-650), as well as proteins with smaller NCMR deletions, exhibit wild-type levels of autophosphorylation and kinase activity (B and C and data not shown; summarized in ). Apparently, no specific sequence in the NCMR has an essential role.
We also tested the biological function of the truncated Gwl proteins in Xenopus
egg extracts. Depletion of endogenous Gwl from CSF extracts leads to a loss of mitotic status that can be prevented by adding active exogenous Gwl (45
). The results in A show the dynamic range of this functional assay. All deletion mutant Gwl proteins just described (made in OA-treated cells) can maintain Gwl-depleted CSF extracts in M phase (D), verifying their biological activity.
Another indication that the NCMR's particular amino acid sequence is nonessential is the finding that Drosophila
Gwl, which has no sequence homology with frog Gwl in the NCMR, can substitute for the Xenopus
enzyme in CSF extracts (data not shown). Fly and frog Gwl are thus functionally orthologous, a conclusion earlier implied by phenotypic similarities (44
Gwl's autophosphorylation is less pronounced than that of Xenopus
Gwl (B), suggesting that some autophosphorylation sites in the frog enzyme are nonessential.
The NCMR is still likely to be important for Gwl regulation, even if particular NCMR sequences are not. For example, Gwl(Δ241-699), in which almost the entire NCMR is removed (), is inactive in both the kinase and extract assays (B to D). Interestingly, however, interphase Gwl with half the NCMR deleted is more easily activated in extracts than the WT protein (B, right). We suggest possible explanations for these paradoxical results in Discussion.
Mutagenesis of potential phosphorylation sites.
To investigate how phosphorylation mitotically activates Gwl, we mutated individually all conserved S and T residues outside the NCMR to nonphosphorylatable A. We altered selected MS-validated sites within the NCMR to serve as controls, but we did not comprehensively analyze all NCMR phosphosites because proteins collectively lacking almost the whole NCMR remain functional. Gwl's mitotic regulation must therefore rely on phosphorylations elsewhere, either within the kinase domain or in the short conserved regions adjacent to the kinase domain (blue in ) that distinguish Gwl from other AGC kinases.
We tested 33 sites by preparing mutant proteins from OA-treated Sf9 cells and analyzing their kinase activities in vitro (A to D). The Gwl mutants with mutations S39A, S89A, S98A, S101A, S119A, T193A, T206A, S212A, T748A, T868A, and S883A exhibited significantly reduced autophosphorylation and kinase activities toward endosulfine or MBP substrates. Among this set of 33 mutants, a linear correlation exists between the phosphorylation of exogenous substrates and of Gwl itself (r = 0.892, P < 1 × 10−6), verifying that Gwl labeling represents autophosphorylation rather than a contaminating kinase (D). The regression line's slope is less than 1, probably because a few mutations, such as S98A and S119A, disrupt substrate recognition but not intrinsic enzyme activity.
Fig 8 Analysis of S/T-to-A mutations. (A) Summary showing specific kinase activities of S/T-to-A mutants expressed in OA-treated cells. Sites marked with asterisks were observed by MS to be phosphorylated in M phase. Sites in boldface are required for Gwl function (more ...)
We also tested the mutant proteins for biological activity in extracts (E and data not shown). The results of these functional analyses were in general consistent with those of the in vitro kinase assays. With the exception of the protein with the S119A mutation, all of the mutant proteins with disrupted kinase activity were also unable to sustain M phase in Gwl-depleted CSF extracts. S119A is probably a borderline case in which the mutant enzyme's reduced activity is still sufficient to cross the threshold needed to sustain M phase in the extracts, between 25 and 50% of the activity of endogenous WT Gwl (A).
We also altered 9 potentially important sites to D or E to mimic phosphorylated S or T, respectively. When expressed in OA-treated cells, most of these mutants had reduced activity in vitro, with the exception of the hyperactive S101D (A and B). Importantly, however, proteins containing the T193E, T206E, and S883D mutations, which are phosphomimetic mutations of the sites most critical to Gwl regulation (see below), are functional in extracts (C) and have only mild reductions in kinase activity in vitro (A and B). The inactivity of the corresponding original T193A, T206A, and S883A mutants thus probably reflects the inability of these sites to be phosphorylated rather than specific structural requirements for T or S at these positions.
Fig 9 Phosphomimetic mutations. (A to C) S/T residues of importance were mutated to D/E, and the proteins expressed in OA-treated Sf9 cells. Mutants were assayed for kinase activity (A and B) and function in CSF extracts (C) as described for . (D to F) (more ...)
Because one purpose of making the phosphomimetic mutants was the generation of constitutively active Gwl, we also expressed combinations of these mutations in Sf9 cells not treated with OA. None of the many variations we tried exhibited full constitutive activity. However, the interphase S101D T193E double mutant has about 10 to 15% of M phase WT Gwl's kinase activity (normalized for protein amount), even though this double mutant remains hypophosphorylated (D). The S101D T193E mutant made in the absence of OA cannot rescue the M phase status of Gwl-depleted CSF extracts except in considerable excess (E). The limited constitutive activity of this mutant enzyme is stimulated in vitro by CDK treatment (data not shown); CDK targeting of sites other than S101 and T193 is thus needed for full Gwl activation.
Mass spectrometry of mitotic phosphorylation sites.
We conducted several MS experiments to identify the M phase phosphosites in frog and fly Gwl. In some experiments, endogenous mitotic Gwl was immunoprecipitated from Xenopus CSF extracts; in others, recombinant Gwl was prepared by expression in OA-treated Sf9 cells. Finally, because CDKs can activate Gwl in vitro (), recombinant Gwl expressed in Sf9 cells in the absence of OA (and thus lacking M phase phosphorylations) was treated with MPF or Cdk2/cyclin A.
Gwl prepared by these methods (C) was digested with trypsin or, to increase coverage, with chymotrypsin or endopeptidase GluC. MS was used to find phosphorylated peptides (). Peptides representing 89% of Gwl were seen at least once, although individual experiments covered only 45 to 80% of the protein. MS identified 23 phosphoamino acids in fly Gwl and >50 phosphoamino acids in frog Gwl, of which ~25 were seen consistently. These numbers roughly correspond with the number of phosphopeptides seen by two-dimensional tryptic mapping (not shown), indicating that MS accounts for most M phase phosphorylation sites. With the exception of phosphotyrosine at positions 727 and 751 in Xenopus
Gwl, all of the phosphorylated sites are S or T. More than half are proline directed, that is, S/T P motifs probably targeted by CDKs. Except for T725, all of the phosphosites we identified in conserved parts of Gwl (that is, outside the NCMR) were previously documented as M phase-specific sites in previous studies of human cells (7
) (results summarized in ). Phosphorylations of Y727 or Y751 are unlikely to play critical roles in Gwl regulation, because fly Gwl has F at the Y727 cognate position, while all AGC kinases have Y at the 751 cognate position.
Only a small subset of MS-detected phosphorylations intersects with the critical phosphorylation sites identified by the mutagenesis experiments described above; these are phosphorylated T193 (pT193), pT206, pS212, pT748, and pS883 in Xenopus
Gwl. We estimated the phosphate occupancy of these sites by comparing the peak areas for all phosphorylated and nonphosphorylated forms of the corresponding peptides (). The phosphate occupancy of all of these sites is higher in active frog Gwl from OA-treated cells than in inactive Gwl from untreated cells. T193 and S883 are likely nearly stoichiometrically occupied by phosphate in active samples; the results presented in probably underestimate occupancies because MS detection efficiencies for phosphopeptides are often >10 times lower than for nonphosphorylated forms (12
). The phosphate occupancy of pT206 also appears to be relatively high (>25%), while those for pS212 and pT748 are more modest. Similar results were observed for occupancy of the corresponding sites in Drosophila
Gwl made in OA-treated cells ().
Occupancy of key phosphorylation sites
Kinases targeting critical phosphorylation sites.
T193 and T206 are proline-directed sites that are probably recognized by CDKs. Indeed, MPF can target both sites in vitro when either a fragment of Gwl containing these sites (A) or full-length inactive Gwl () is used as a substrate. A fragment containing T193 but with the T206A mutation is a better substrate than a fragment with T193A and T206, indicating that T193 is more efficiently targeted by MPF (A).
S883 is not proline directed, and a fragment including this site is not phosphorylated in vitro
by MPF (A). Indeed, the region surrounding S883 does not conform to the consensus sequence for any known kinase. We thus tested the idea that Gwl itself can target S883 through autophosphorylation. As shown in , pS883 is produced when inactive WT frog Gwl (made in the absence of OA) is partially activated in vitro
with MPF but not when KD Gwl is used instead. Furthermore, WT Gwl (but not KD Gwl, Plx1, or Cdk1/cyclin A) can utilize a short peptide containing S883 as an exogenous substrate (B), and MS analysis demonstrates that pS883 is the only site Gwl phosphorylates in this peptide (data not shown). The S883-containing peptide is phosphorylated ~200-fold less efficiently than a similarly sized peptide containing the Gwl target site in endosulfine (data not shown), consistent with experiments on other kinases using peptides containing autophosphorylation sites as exogenous substrates (11