AMP-activated protein kinase–mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β

The Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)/5′-AMP–activated protein kinase (AMPK) phosphorylation cascade affects various Ca2+-dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca2+ concentrations. Moreover, the Ca2+/CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro, leading to reduced autonomous, but not Ca2+/CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr144 phosphorylation by activated AMPK converts CaMKKβ into a Ca2+/CaM-dependent enzyme as shown by completely Ca2+/CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471–587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr144 phosphorylation–dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr144 antibody revealed phosphorylation of Thr144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca2+-dependent AMPK activation by CaMKKβ.

domain (Thr 108 ) and CaM-binding domain (Ser 458 ) of CaMKK␣, thus facilitating the recruitment of 14-3-3 protein and suppression of CaMKK activity in vivo and in vitro (35)(36)(37)(38). In contrast to CaMKK␣, recombinant CaMKK␤ exhibits a higher basal activity (i.e. in the absence of Ca 2ϩ /CaM) (6,39). This is partly attributed to intramolecular autophosphorylation at Thr 482 , resulting in partial disruption of the autoinhibitory mechanism (40). In addition, the N-terminal regulatory region (residues 129 -151) was found to affect the autonomous activity of rat CaMKK␤ as the deletion of this domain conferred Ca 2ϩ /CaM dependence on the kinase (39).
Beyond autophosphorylation, cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3 (GSK3) can phosphorylate multiple residues in the N-terminal regulatory domain (Ser 129 , Ser 133 , and Ser 137 in human CaMKK␤), resulting in decreased autonomous activity (41). This observation is in agreement with the finding that CaMKK␤/AMPK pathway activation requires Ca 2ϩ /CaM signaling, whereas the CaMKK␤ substrate AMPK is not Ca 2ϩ /CaM-dependent (13)(14)(15)32). According to those studies, the maintenance of CaMKK␤ as a Ca 2ϩ /CaM-dependent form appears to depend on multiple phosphorylation events, including autophosphorylation and the effects of other protein kinases. Because the effects of closely proximal downstream kinases on the activity of CaMKK␤ have not yet been evaluated, we attempted to examine this activity during CaMKK␤-mediated AMPK activation. Here, we observed that, in vitro, phosphorylation by CaMKK␤-activated AMPK significantly decreased autonomous CaMKK␤ activity. We also identified a single AMPK phosphorylation site in the N-terminal regulatory domain of CaMKK␤ that acts as a switch for Ca 2ϩ / CaM dependence and suggests a unique enzymatic mechanism of CaMKK␤/AMPK signaling cascade regulation.

Autonomous activity of recombinant CaMKK␤ is suppressed during AMPK activation
Although recombinant CaMKK␤ has been shown to exhibit a higher basal activity in the absence of Ca 2ϩ /CaM (autonomous activity) (6,39), in intact cells CaMKK␤ signaling activation requires an increased intracellular Ca 2ϩ concentration (13)(14)(15)32). Therefore, we first examined the activity of Escherichia coli-expressed CaMKK␤ during a CaMKK␤-mediated AMPK activation reaction in which we incubated CaMKK␤ with either wild-type or kinase-dead mutant (K45R) AMPK in the presence of Mg-ATP and EGTA for various time points (5-60 min) followed by the withdrawal of constant amounts of reaction mixture into buffer containing excess EDTA to stop the phosphorylation reaction. Subsequently, CaMKK␤ activity levels in the samples were measured by a 10-min kinase reaction assay with glutathione S-transferase (GST)-tagged CaMKI␣(1-293) K49E as the substrate in the absence of Ca 2ϩ / CaM (autonomous activity). When 100 M [␥-32 P]ATP was used (Fig. 1A), CaMKK␤ autonomous activity decreased gradually in the presence of wild-type AMPK but was not affected by incubation with AMPK K45R mutant. To confirm that GST-CaMKI␣(1-293) K49E phosphorylation was mediated by CaMKK␤ at Thr 177 (CaMKK phosphorylation site), we con-ducted a 5-min kinase reaction assay using non-radioisotopic ATP (Fig. 1B) followed by a dot-blotting assay and antibody-mediated detection of Thr 177 phosphorylation in GST-CaMKI␣(1-293) K49E (Fig. 1B, inset). The similar results obtained from these two different CaMKK activity assays confirmed the suppression of CaMKK␤ autonomous activity by  (B; dot-blot  assay). B, inset, shows results of one set of dot-blot assays using anti-phospho-CaMKI (at Thr 177 ) antibody. An arrow indicates the no-enzyme control. Autonomous activities of CaMKK␤ in B are expressed as a percentage of the average value at 0 min, and the results represent two sets of dot-blot assays. C, reaction mixtures (50 ng of CaMKK␤) as shown in B were subjected to SDS-7.5% PAGE followed by immunoblot analysis using an anti-CaMKK antibody. The molecular mass in kilodaltons is indicated on the left. D, recombinant CaMKK␤ (1.2 g) was incubated without (Ϫ) or with WT AMPK (1.2 g) at 30°C for 60 min in a solution (20 l) containing 50 mM HEPES, pH 7.5, 10 mM Mg(CH 3 COO) 2 , and 1 mM DTT in the presence of 2 mM EGTA either without (Ϫ) or with (ϩ) 1 mM ATP, and then the reaction was terminated followed by measuring CaMKK␤ activity using a dot-blot assay as described in B. Autonomous activities of CaMKK␤ are expressed as a percentage of the average value in the absence of AMPK. E, recombinant CaMKK␤ (1.2 g) was incubated without (Ϫ) or with either WT or K45R mutant AMPK (1.2 g) at 30°C for 60 min in a solution (20 l) containing 50 mM HEPES, pH 7.5, 10 mM Mg(CH 3 COO) 2 , and 1 mM DTT in the presence of 2 mM EGTA and 1 mM ATP, and then the reaction was terminated followed by measuring CaMKK␤ activity using 100 M [␥-32 P]ATP in the presence of 2 mM EGTA (Ϫ) (white bars) or 2 mM CaCl 2 and 6 M CaM (ϩ) (black bars). Results in A, D, and E are expressed as the mean Ϯ S.D. of three experiments. Error bars represent S.D. Statistical differences are marked: *, p Ͻ 0.05, **, p Ͻ 0.01, n.s., not significant.

Feedback phosphorylation of CaMKK␤ by AMPK
wild-type AMPK. We confirmed that AMPK␣ subunit was activated through phosphorylation at Thr 172 by CaMKK␤ during the incubation periods (see supplemental Fig. S1).
When samples collected at various time points (0 -60 min) as shown in Fig. 1B were analyzed by immunoblotting using an anti-CaMKK antibody (Fig. 1C), the electrophoretic mobility of the CaMKK␤ on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gradually shifted upward in the presence of wild-type AMPK but not K45R mutant enzyme, suggesting that this shift was mediated by AMPK-catalyzed phosphorylation. When we incubated wild-type AMPK and CaMKK␤ without ATP, CaMKK␤ autonomous activity decreased slightly (ϳ25%) relative to the 90% reduction observed in the presence of 1 mM ATP, indicating that the CaMKK␤-mediated, phosphorylation-dependent activation of AMPK is required for the subsequent inhibition of CaMKK␤ activity by activated AMPK (Fig. 1D). The slight reduction in CaMKK␤ activity in the absence of ATP was likely due to the phosphorylation of CaMKK␤ by activated AMPK during the subsequent CaMKK␤ activity assay. These results suggest that, following CaMKK␤-mediated phosphorylation, activated AMPK subsequently phosphorylates CaMKK␤ to suppress its autonomous activity.

CaMKK␤ catalytic activity is not affected by AMPK phosphorylation
To clarify the molecular mechanism by which activated AMPK suppresses CaMKK␤ autonomous activity, we produced unphosphorylated (without AMPK or with AMPK K45R mutant) and phosphorylated CaMKK␤ (with activated AMPK) and measured the Ca 2ϩ /CaM dependence of these enzymes (Fig. 1E). Consistent with Fig. 1, A and B, AMPK-mediated phosphorylation significantly suppressed the basal activity (autonomous activity) of recombinant CaMKK␤ but did not affect the total activity in the presence of Ca 2ϩ /CaM. In other words, phosphorylation by AMPK was unable to suppress CaMKK␤ catalytic activity but could convert the enzyme into a Ca 2ϩ / CaM-dependent kinase. We also performed a time-course experiment to confirm that the total activity of CaMKK␤ was not altered by incubation with AMPK in the presence of Ca 2ϩ / CaM (supplemental Fig. S3).

AMPK phosphorylation of CaMKK␤ Thr 144 is involved in the suppression of autonomous activity
We next attempted to identify both autophosphorylation and AMPK phosphorylation sites in CaMKK␤ via liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to determine which were involved in the suppression of autonomous activity. In addition to seven autophosphorylation sites, we identified multiple AMPK phosphorylation sites, including Ser 110 , Ser 126 , Ser 135 , Thr 144 , Ser 174 , Thr 446 , Ser 494 , Ser 510 , and Ser 552 , in recombinant CaMKK␤ incubated with K45R (autophosphorylation) or wild-type AMPK (autophosphorylation plus AMPK phosphorylation) in the presence of Mg-ATP at 30°C for 60 min (Table 1, Fig. 2A, and supplemental Fig. S2). Because AMPK-catalyzed phosphorylation significantly reduced the Ca 2ϩ /CaM-independent activity (autonomous activity) of CaMKK␤ without affecting its catalytic activity ( Fig.  1), we first constructed three Ala-substituted mutants (S135A, T144A, and S494A) in which the phosphorylation sites were located either in the N-terminal regulatory region (residues 129 -151) or the C-terminal autoinhibitory domain (AID) (residues 474 -499). After expression and purification, these mutant CaMKK␤s were evaluated to determine the effects of AMPK on autonomous activity (Fig. 2B). Whereas AMPK suppressed the autonomous activities of the S135A and S494A mutants in a manner similar to the suppression of wild-type CaMKK␤ (Fig. 2B), the T144A mutant was unaffected, indicating that CaMKK␤ autonomous activity is suppressed by AMPK through direct phosphorylation at Thr 144 . This conclusion was confirmed by the finding that a phosphomimetic mutant (T144E) of CaMKK␤ was a completely Ca 2ϩ /CaM-dependent enzyme and exhibited no significant autonomous activity ( Fig.  2C) compared with wild type (70 -80% of total activity) and T144A CaMKK␤ (ϳ50% of total activity). We measured specific activities in the presence of Ca 2ϩ /CaM of all the point mutants (S135A, 580 Ϯ 31 nmol/min/mg; T144A, 390 Ϯ 20 nmol/min/mg; S494A, 394 Ϯ 18 nmol/min/mg; and T144E, Table 1 Identification

of autophosphorylation and AMPK phosphorylation sites in rat CaMKK␤
Recombinant rat CaMKK␤ (E. coli) was phosphorylated by either wild-type or kinase-dead mutant (K45R) AMPK for 60 min followed by LC-MS/MS analysis to identify the autophosphorylation (Autophos. site) and AMPK phosphorylation sites (AMPK site) as described under "Experimental procedures" (see supplemental Fig. S2).

Phosphorylation
Residues Peptide sequence

Phospho-amino acid residues
Autophos. site 14 a Autophosphorylation sites of rat CaMKK␤ were identified previously (40). b Autophosphorylation of Thr 517 was identified in rat CaMKK␤ incubated with AMPK K45R mutant.

Feedback phosphorylation of CaMKK␤ by AMPK
529 Ϯ 16 nmol/min/mg) and wild-type CaMKK␤ (515 Ϯ 42 nmol/min/mg) using a 5-min standard CaMKK activity assay as described under "Experimental procedures," indicating that the mutant CaMKK␤s have total activities comparable with that of wild-type enzyme.
In addition, we expressed CaMKK␤, including wild type and Thr 144 mutants, in COS-7 cells and measured CaMKK activities using an equal volume of cell lysates (1 l) (Fig. 2D). Whereas total activities (in the presence of Ca 2ϩ /CaM) of exogenously expressed CaMKK␤, including wild type (7.8 Ϯ 1.9 pmol/min/l of lysate), T144A mutant (7.8 Ϯ 0.3 pmol/min/l of lysate), and T144E mutant (7.9 Ϯ 0.3 pmol/min/l of lysate), are indistinguishable, we observed significant autonomous activity (ϳ40% of total activity) of wild-type enzyme expressed in COS-7 cells that was consistent with a previous report (39), and the Glu mutation significantly reduced the basal activity (ϳ10% of total activity). This is in good agreement with the results using E. coli-expressed CaMKK␤ as shown in Fig. 2C.

Phosphorylation of CaMKK␤ at Thr 144 in vitro and in living cells
To confirm the physiologic significance of Thr 144 phosphorylation of CaMKK␤, we generated a monoclonal antibody that specifically recognized a phosphorylated form of CaMKK␤ at Thr 144 as described under "Experimental procedures." Immunoblot analysis (Fig. 3A, top panel) revealed that the antibody recognized CaMKK␤ phosphorylated with wild-type AMPK but not an autophosphorylated CaMKK␤. In addition, CaMKK␤ mutant T144A phosphorylated with wild-type AMPK was not recognized by the antibody, indicating that the antibody is capable of specifically recognizing the Thr 144 -phosphorylated form of CaMKK␤. Then we analyzed cell lysates of COS-7 cells transfected with rat CaMKK␤ expression plasmid with or without HA-AMPK␣ expression plasmid by immunoblotting using the anti-phospho-Thr 144 antibody. Fig. 3B (top and middle panels) clearly shows that the exogenously expressed CaMKK␤ in COS-7 cells was phosphorylated at Thr 144 , and this phosphorylation was induced 3-7-fold by coexpression of HA-AMPK␣, indicating the AMPK-mediated phosphorylation of CaMKK␤ at Thr 144 in living cells as well as in vitro. We confirmed the exogenous expression of HA-AMPK␣ in the cells (Fig. 3B, bottom panel) and that T144A mutant coexpressed with HA-AMPK␣ was not detected by the immunoblot analysis using the anti-phospho-Thr 144 antibody (Fig. 3B, top panel) in good agreement with the result shown in Fig. 3A.
To test whether other CaMKK target kinases are capable of phosphorylating the Thr 144 in CaMKK␤, we prepared CaMKK␤ samples incubated with its downstream protein kinases, including GST-rat CaMKI␣ and GST-mouse CaM-KIV, in the presence of Ca 2ϩ /CaM and Mg-ATP in vitro (Fig.  3C) followed by immunoblot analyses. Both downstream CaMKs were capable of phosphorylating CaMKK␤ at Thr 144 but less efficiently than AMPK (Fig. 3C, top panel). We confirmed that both downstream CaMKs were activated by phosphorylation of their activation-loop Thr residues (Thr 177 in CaMKI and Thr 196 in CaMKIV) with CaMKK␤ during the incubation process in a similar manner to AMPK phosphoryla-

Feedback phosphorylation of CaMKK␤ by AMPK
tion at Thr 172 (Fig. 3C, middle panel), indicating that the feedback phosphorylation of CaMKK␤ at Thr 144 was catalyzed by activated CaMKK target kinases.

Phosphorylation-dependent reduction of CaMKK␤ autonomous activity involves the C terminus
Our data, which suggest that Thr 144 phosphorylation converts CaMKK␤ to a Ca 2ϩ /CaM-dependent form, are in good agreement with many previous studies in which CaMKK␤ activity was shown to require increases in intracellular Ca 2ϩ concentration (13)(14)(15)32). We next attempted to elucidate the molecular mechanism underlying the effect of Thr 144 phosphorylation on the enzymatic regulation of CaMKK␤ by using a C-terminal truncation mutant (Fig. 4A) that had been phosphorylated or not by AMPK. A CaMKK␤(2-470) molecule that lacked the C-terminal regulatory domain (Val 474 -Phe 499 ), which contains an AID and CaM-binding region, was constitutively active (39). We confirmed that both the wild-type and CaMKK␤(2-470) mutant enzymes were phosphorylated at Thr 144 by AMPK (Fig. 4C). In contrast to wild-type CaMKK␤ whose basal activity was significantly reduced by AMPK-catalyzed phosphorylation, the C-terminal truncation mutant CaMKK␤(2-470) did not respond to the Thr 144 phosphorylation by AMPK (Fig. 4B), indicating that the C-terminal region, including the AID (residues 474 -499), plays an important Thr 144 phosphorylation-dependent role in constructing an inactive conformation. This result also confirmed that the phosphorylation by AMPK was unable to suppress CaMKK␤ total

Discussion
Our biochemical data demonstrate that the autonomous activity (in the absence of Ca 2ϩ /CaM) of CaMKK␤ is reduced by an activated downstream kinase, AMPK, via feedback phosphorylation at Thr 144 . Thr 144 phosphorylation converts CaMKK␤ into a Ca 2ϩ /CaM-dependent enzyme, corroborating previous studies in which the activation of CaMKK␤-mediated signaling cascades, including AMPK, CaMKI, and CaMKIV pathways, was found to depend on increasing concentrations of intracellular Ca 2ϩ (Fig. 5) (13-15, 32). This is a unique phosphorylation-dependent regulatory mechanism among CaMKs. CaMKII is known to undergo autophosphorylation at Thr 286 (␣-subunit within the AID) and thus induce autonomous activity (42)(43)(44). In the case of CaMKIV, Thr 196 phosphorylation by CaMKK reduces the interaction of the catalytic core with the AID, resulting in generation of the autonomous activity (45). Unlike CaMKK␣ expressed in E. coli or COS-7 cells, which was found to be completely Ca 2ϩ /CaM-dependent kinase due to an autoinhibitory mechanism (46), CaMKK␤ exhibits significant autonomous activity (70 -80% of total activity), resulting from the impairment of a regulatory domain (residues 474 -499)mediated autoinhibitory mechanism (6,39).
Previously, we have identified involvement of the N-terminal regulatory domain (residues 129 -151) in rat CaMKK␤ autonomous activity as deletion of this domain converts CaMKK␤ into a Ca 2ϩ /CaM-dependent kinase (39). Rat CaMKK␤ expressed in E. coli is highly autophosphorylated, and the Ca 2ϩ / CaM-independent, intramolecular phosphorylation of Thr 482 in the AID (residues 474 -499) was also detected in the present study (Table 1 and supplemental Fig. S2). Thr 482 autophosphorylation, together with the N-terminal regulatory domain, results in Ca 2ϩ /CaM-independent, or basal, activity (Fig. 5, Autonomous Activity) (40). In this report, we found that the phosphorylation by AMPK at Thr 144 , within the N-terminal regulatory domain (residues 129 -151), largely reduced the basal activity of CaMKK␤ without significantly affecting the catalytic activity ( Fig. 1E and Fig. 5, Inactive). This suggests that Thr 144 phosphorylation disrupts the function of the N-terminal regulatory domain, which is necessary for autonomous activity (39), thus converting CaMKK␤ into a Ca 2ϩ /CaM-dependent enzyme (Fig. 5, Ca 2ϩ /CaM-dependent Activity). This suggestion was confirmed by the finding that the enzymatic activity of a CaMKK␤ mutant containing a single Thr 144 phosphomimetic (Glu) mutation was entirely Ca 2ϩ /CaM-dependent (Fig. 2, C  and D). Our observations agree well with an analogous mechanism demonstrated by Green et al. (41) in which the phosphorylation of multiple residues (Ser 129 , Ser 133 , and Ser 137 ) in the N-terminal regulatory domain of human CaMKK␤ by CDK5 and GSK3 led to decreased autonomous activity.
Notably, the CDK5 and GSK3 phosphorylation sites (Ser 128 , Ser 132 , and Ser 136 ) in rat CaMKK␤ were not phosphorylated by activated AMPK in vitro (Table 1). Our results suggest that the regulation of CaMKK␤ by phosphorylation via activated AMPK might be physiologically relevant because AMPK is a direct downstream kinase in close proximity to CaMKK␤. This is confirmed by the fact that the exogenously expressed  (Table 1), resulting in generation of the autonomous activity together with the function of the N-terminal regulatory domain (Autonomous Activity) (40). CaMKK␤ phosphorylates the ␣-subunit of AMPK at Thr 172 to activate its catalytic activity. Activated AMPK phosphorylates CaMKK␤ at Thr 144 to convert the enzyme to its inactive form in the absence of Ca 2ϩ /CaM (Inactive). Phosphorylated CaMKK␤ is fully activated by Ca 2ϩ /CaM binding (Ca 2ϩ /CaM-dependent Activity) to phosphorylate and activate downstream protein kinases, including CaMKI, CaMKIV, and AMPK. N, N-terminal regulatory domain (residues 129 -151) (39); Catalytic, catalytic domain (residues 162-470); AID, autoinhibitory domain containing the Ca 2ϩ /CaMbinding region (residues 474 -499) (46); ␣/␤/␥, ␣-subunit/␤-subunit/␥-subunit of AMPK; CaMKI⅐KIV, Ca 2ϩ /CaM-dependent protein kinases I and IV; T, Thr residue. P in a black box indicates phosphorylation.

Feedback phosphorylation of CaMKK␤ by AMPK
CaMKK␤ in cultured cells has been shown to be phosphorylated at Thr 144 , and this phosphorylation was further enhanced by coexpression of AMPK␣ (Fig. 3B). We also observed significant Ca 2ϩ /CaM dependence of the T144E mutant CaMKK␤ expressed in COS-7 cells (Fig. 2D). However, we cannot exclude the possibility that Thr 144 phosphorylation is mediated by other downstream protein kinases, including CaMKI, CaMKIV, and another unidentified cellular protein kinase, because an equivalent Thr residue (Thr 108 ) in CaMKK␣ has been shown to be phosphorylated by cAMP-dependent protein kinase (35,36). Therefore, we tested the Thr 144 phosphorylation in CaMKK␤ by downstream kinases, including CaMKI and CaMKIV, in vitro (Fig. 3C). Immunoblot analysis revealed that both CaMKI and CaMKIV activated by CaMKK␤-mediated phosphorylation are capable of feedback-phosphorylating Thr 144 in CaMKK␤ but less efficiently than the activated AMPK. It would be interesting to determine whether Thr 144 phosphorylation of CaMKK␤ is regulated in a cellular context. Although the N-terminal regulatory domain containing Thr 144 (in rat CaMKK␤) is conserved in various mammalian species (5, 6), a detailed mechanism underlying the release of the AID from the catalytic core by the N-terminal regulatory domain, thus resulting in the generation of autonomous activity, remains unclear (39). However, when we deleted the C-terminal region (residues 471-587), including the AID, of CaMKK␤, the autonomous activity of the mutant (CaMKK␤(2-470)) was no longer affected by AMPK-catalyzed Thr 144 phosphorylation, even in the presence of the N-terminal regulatory domain, suggesting that the C-terminal region, including the AID, is involved in the Thr 144 phosphorylation-dependent reduction of the autonomous activity (Fig. 4). We propose that the N-terminal regulatory region (residues 129 -151) interacts with the C-terminal region (residues 471-587) in a Thr 144 phosphorylation-dependent manner (Fig. 5, Inactive), thus promoting the interaction of the catalytic domain (residues 162-470) with the AID (residues 474 -499). As intracellular Ca 2ϩ levels increase in response to extracellular stimulation, Ca 2ϩ /CaM binds to the C terminus of AID and disrupts the interaction of the catalytic domain with AID to generate CaMKK␤ kinase activity (Fig. 5, Ca 2ϩ /CaM-dependent Activity) (46), thus constituting a Ca 2ϩ -dependent signal transduction pathway. Further study is needed to verify this hypothesis and clarify how the phosphorylation of a single Thr 144 would promote the autoinhibition of CaMKK␤ and thus generate Ca 2ϩ /CaM dependence.

AMPK phosphorylation of CaMKK␤
Recombinant CaMKK␤ (1.2 g) was incubated without or with downstream protein kinases (1.2 g), including wild-type AMPK, K45R mutant, GST-CaMKI␣, and GST-CaMKIV, at 30°C for the indicated time periods in a solution (20 l) containing 50 mM HEPES, pH 7.5, 10 mM Mg(CH 3 COO) 2 , 1 mM DTT, and 1 mM ATP in the presence of 2 mM EGTA or 2 mM CaCl 2 and 6 M CaM. Reactions were terminated by a 25-fold dilution in ice-cold dilution buffer (50 mM HEPES, pH 7.5, 2 mg/ml bovine serum albumin, 10% ethylene glycol, and 2 mM EDTA) for the CaMKK activity assay or the addition of 5 or 20 l of 2ϫ SDS-PAGE sample buffer for either mass spectrometry or immunoblot analysis.

Mass spectrometry analysis
Recombinant rat CaMKK␤ (1.2 g) was incubated with either wild-type or K45R AMPK (1.2 g) in the presence of 1 mM ATP at 30°C for 60 min as described above after which the reaction was terminated by adding 5 l of 2ϫ SDS-PAGE sample buffer. Phosphorylated CaMKK␤ was separated by 7.5% SDS-PAGE and lightly stained with Coomassie Brilliant Blue followed by in-gel digestion (50) with a protease or protease mixture, including trypsin, chymotrypsin (Roche Diagnostics), trypsin with chymotrypsin, trypsin with Glu-C (Roche Diagnostics), trypsin with Asp-N (Roche Diagnostics), or chymotrypsin with Asp-N. The following protease concentrations were used: 10 g/ml trypsin, 17 g/ml chymotrypsin, 10 g/ml Glu-C, and 4 g/ml Asp-N. Trypsin and Asp-N were incubated at 37°C, and chymotrypsin and Glu-C were incubated at 25°C. The first and second digestions were incubated overnight and for 3 h, respectively. The digested peptides were eluted with 0.1% formic acid and subjected to LC-MS/MS analysis on an LC-MS-IT-TOF instrument (Shimadzu, Kyoto, Japan) interfaced with a nano reverse-phase LC system (Shimadzu) as described previously (40). MS/MS data were acquired in the datum-dependent mode using LC-MS solution software (Shimadzu) and converted to a single text file (containing the observed precursor peptide m/z, fragment ion m/z, and intensity values) using Mascot Distiller (Matrix Science, London, UK). MS/MS data were obtained independently and merged for the Mascot analysis. The following search parameters were used: database, rat CaMKK␤ (578 amino acid residues); enzyme, all; variable modifications, carbamidomethyl (Cys), oxidation (Met), propionamide (Cys), and phospho (Ser/Thr).

Other methods
Immunoblot and dot-blot analyses were performed with the indicated primary antibodies and horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (GE Healthcare) as the secondary antibody. A chemiluminescent reagent (PerkinElmer Life Sciences) was used for signal detection of immunoblots followed by quantification of the immunoreactivity with ImageJ software (51). Protein concentrations in samples were estimated by Coomassie Brilliant Blue (Bio-Rad) using bovine serum albumin as a standard. Student's t tests were used to evaluate the statistical significance of two-group comparisons. Probability (p) values Ͻ0.05 were considered statistically significant.