Mechanism of the Generation of Autonomous Activity of Ca2+/Calmodulin-dependent Protein Kinase IV

Tokumitsu, Hiroshi; Hatano, Naoya; Inuzuka, Hiroyuki; Yokokura, Shigeyuki; Nozaki, Naohito; Kobayashi, Ryōji

doi:10.1074/jbc.m406534200

Cited by 49 publications

(51 citation statements)

References 48 publications

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“…However, we currently believe that this is because of the complex structural/functional properties of CaMKK␣, CaMKI, and CaMK phosphatase, and not because CaMKK␣ and CaMKI are inactive during muscle contraction. Directly assessing CaMKK␣ or CaMKI under physiological conditions is extremely challenging because of the lack of Ca 2ϩ /calmodulin-independent activity of these enzymes (39,40). Thus, immediately after the removal of a stimulus, and a decline in intracellular Ca 2ϩ levels, CaMKK␣ is not able to maintain kinase activity (40) and stops phosphorylating CaMKI.…”

mentioning

confidence: 99%

Ca2+/Calmodulin-Dependent Protein Kinase Kinase-α Regulates Skeletal Muscle Glucose Uptake Independent of AMP-Activated Protein Kinase and Akt Activation

et al. 2007

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Studies in nonmuscle cells have demonstrated that Ca2؉ / calmodulin-dependent protein kinase kinases (CaMKKs) are upstream regulators of AMP-activated protein kinase (AMPK) and Akt. In skeletal muscle, activation of AMPK and Akt has been implicated in the regulation of glucose uptake. The objective of this study was to determine whether CaMKK␣ regulates skeletal muscle glucose uptake, and whether it is dependent on AMPK and/or Akt activation. Expression vectors containing constitutively active CaMKK␣ (caCaMKK␣) or empty vector were transfected into mouse muscles by in vivo electroporation. After 2 weeks, caCaMKK␣ was robustly expressed and increased CaMKI (Thr 177/180 ) phosphorylation, a known CaMKK substrate. In muscles from wild-type mice, caCaMKK␣ increased in vivo [ 3 H]-2-deoxyglucose uptake 2.5-fold and AMPK␣1 and -␣2 activities 2.5-fold. However, in muscles from AMPK␣2 inactive mice (AMPK␣2i), caCaMKK␣ did not increase AMPK␣1 or -␣2 activities, but it did increase glucose uptake 2.5-fold, demonstrating that caCaMKK␣ stimulates glucose uptake independent of AMPK. Akt (Thr 308 ) phosphorylation was not altered by CaMKK␣, and caCaMKK␣ plus insulin stimulation did not increase the insulin-induced phosphorylation of Akt (Thr 308 ). These results suggest that caCaMKK␣ stimulates glucose uptake via insulin-independent signaling mechanisms. To assess the role of CaMKK in contraction-stimulated glucose uptake, isolated muscles were treated with or without the CaMKK inhibitor STO-609 and then electrically stimulated to contract. Contraction increased glucose uptake 3.5-fold in muscles from both wild-type and AMPK␣2i mice, but STO-609 significantly decreased glucose uptake (ϳ24%) only in AMPK␣2i mice. Collectively, these results implicate CaMKK␣ in the regulation of skeletal muscle glucose uptake independent of AMPK and Akt activation. Diabetes 56:1403-1409, 2007 I n the U.S., 90 -95% of all diagnosed cases of diabetes are classified as type 2 diabetes (1), a form of diabetes where target tissues such as skeletal muscle do not respond properly to insulin. In these patients, insulin-dependent signaling mechanisms regulating skeletal muscle glucose uptake are impaired (2). Importantly, insulin-independent mechanisms, including muscle contraction or exercise-mediated mechanisms for regulating glucose uptake, remain intact (3). Thus, elucidation of the signaling pathways governing contractioninduced increases in skeletal muscle glucose uptake may provide new pharmacological targets for the treatment of individuals with type 2 diabetes.Muscle contraction is a multifactorial process involving changes in energy status (i.e., increased AMP-to-ATP ratio), increases in intracellular Ca 2ϩ levels, stretch, etc., and it is likely that multiple signaling pathways act to increase plasma membrane GLUT4 transporters and glucose uptake. Studies using the AMP analog, 5-aminoimidazole-4-carboxamide-1-␤-D-ribofuranotide (AICAR), have demonstrated that activation of AMP-activated protein kinase (AMPK) is positively correlated with an i...

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confidence: 99%

Ca2+/Calmodulin-Dependent Protein Kinase Kinase-α Regulates Skeletal Muscle Glucose Uptake Independent of AMP-Activated Protein Kinase and Akt Activation

et al. 2007

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“…Calmodulin kinases are classically thought to be regulated by calcium/calmodulin for full activation but are known to have kinase activity independent of calcium/calmodulin, resulting in autophosphorylation (19). In fact, Thr286 and Thr196 phosphorylation, in calmodulin kinase II and IV, respectively, generated an autonomous kinase activity which could be further activated by calcium/calmodulin (4,61). Pnck-induced EGFR degradation is dependent on cell density, and maximal EGFR degradation was observed at low cell density, with degradation gradually being suppressed with increasing cell density.…”

Section: Discussionmentioning

confidence: 94%

Pnck induces ligand-independent EGFR degradation by probable perturbation of the Hsp90 chaperone complex

Deb

Zuo

Wang

et al. 2011

American Journal of Physiology-Cell Physiology

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We have recently described a novel role for pregnancy-upregulated non-ubiquitous calmodulin kinase (Pnck) in the induction of ligand-independent epidermal growth factor receptor (EGFR) degradation (Deb TB, Coticchia CM, Barndt R, Zuo H, Dickson RB, and Johnson MD. Am J Physiol Cell Physiol 295: C365-C377, 2008). In the current communication, we explore the probable mechanism by which Pnck induces ligand-independent EGFR degradation. Pnck-induced EGFR degradation is calcium/calmodulin independent and is regulated by cell density, with the highest EGFR degradation observed at low cell density. Pnck is a novel heat shock protein 90 (Hsp90) client protein that can be co-immunoprecipitated with Hsp90. Treatment of Pnck-overexpressing cells with the pharmacologic Hsp90 inhibitor geldanamycin results in enhanced EGFR degradation, and destruction of Pnck. In cells in which Pnck is inducing EGFR degradation, we observed that Hsp90 exhibits reduced electrophoretic mobility, and through mass spectrometric analysis of immunopurified Hsp90 protein we demonstrated enhanced phosphorylation at threonine 89 and 616 (in both Hsp90-α and -β) and serine 391 (in Hsp90-α). Kinase-active Pnck protein is degraded by the proteasome, concurrent with EGFR degradation. A Pnck mutant (T171A) protein with suppressed kinase activity induced EGFR degradation to essentially the same level as wild-type (WT) Pnck, suggesting that Pnck kinase activity is not required for the induction of EGFR degradation. Although EGFR is degraded, overexpression of WT Pnck paradoxically promoted cellular proliferation, whereas cells expressing mutant Pnck (T171A) were growth inhibited. WT Pnck promoted S to G(2) transition, but cells expressing the mutant exhibited higher residency time in S phase. Basal MAP kinase activity was inhibited by WT Pnck but not by mutant T171A Pnck protein. Cyclin-dependent kinase (Cdk) inhibitor p21/Cip-1/Waf-1 was transcriptionally suppressed downstream to MAP kinase inhibition by WT Pnck, but not the mutant protein. Collectively, these data suggest that 1) Pnck induces ligand-independent EGFR degradation most likely through perturbation of Hsp90 chaperone activity due to Hsp90 phosphorylation, 2) EGFR degradation is coupled to proteasomal degradation of Pnck, and 3) modulation of basal MAP kinase activity, p21/Cip-1/Waf-1 expression, and cellular growth by Pnck is independent of Pnck-induced ligand-independent EGFR degradation.

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“…This GSTCaM-KI CD had a cleavage site (Leu-Glu-Val-Leu-Phe-Gln2Gly-Pro) for PreScission protease between GST and the CaM-KI CD, which led to the specific elution of the CaM-KI CD-interacting proteins from the affinity matrix by protease treatment. GST-fused CaM-KI CD was fully phosphorylated with CaM-KK in the presence of Ca 2ϩ /CaM for 2 h, as was recently demonstrated (32), and then phosphorylated CaM-KI CD was coupled with glutathione-Sepharose resin (Fig. 1A, c).…”

Section: Identification Of Cam-ki Substrates Using a Functional Protementioning

confidence: 96%

“…GST-14-3-3 was expressed in E. coli JM-109, followed by purification by glutathione-Sepharose chromatography (51). Anti-phospho-CaM-KI at Thr 177 monoclonal antibody was generated as previously described (32). Anti-CaM-KI and anti-CaM-KK antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Transduction Laboratories, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The recombinant protein was expressed in Escherichia coli JM-109, followed by purification by glutathione-Sepharose chromatography. Recombinant wild-type rat CaM-KI␣, wild-type mouse CaM-KIV, and wild-type rat CaM-KK␣ were expressed and purified as previously described (31,32). Recombinant rat CaM was expressed in the Epicurian coli BL-21 (DE3) using pET-CaM (kindly provided by Dr. Nobuhiro Hayashi, Fujita Health University, Toyoake, Japan) and was purified by phenyl-Sepharose column chromatography (33).…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation of Numb Family Proteins

Tokumitsu

Hatano

Inuzuka

et al. 2005

Journal of Biological Chemistry

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To search for the substrates of Ca 2؉ /calmodulin-dependent protein kinase I (CaM-KI), we performed affinity chromatography purification using either the unphosphorylated or phosphorylated (at Thr 177 ) GST-fused CaM-KI catalytic domain (residues 1-293, K49E) as the affinity ligand. Proteomic analysis was then carried out to identify the interacting proteins. In addition to the detection of two known CaM-KI substrates (CREB and synapsin I), we identified two Numb family proteins (Numb and Numbl) from rat tissues. These proteins were unphosphorylated and were bound only to the Thr 177 -phosphorylated CaM-KI catalytic domain. This finding is consistent with the results demonstrating that Numb and Numbl were efficiently and stoichiometrically phosphorylated in vitro at equivalent Ser residues (Ser 264 in Numb and Ser 304 in Numbl) by activated CaM-KI and also by two other CaM-Ks (CaM-KII and CaM-KIV). Using anti-phospho-Numb/Numbl antibody, we observed the phosphorylation of Numb family proteins in various rat tissue extracts, and we also detected the ionomycin-induced phosphorylation of endogenous Numb at Ser 264 in COS-7 cells. The present results revealed that the Numb family proteins are phosphorylated in vivo as well as in vitro. Furthermore, we found that the recruitment of 14-3-3 proteins was the functional consequence of the phosphorylation of the Numb family proteins. Interaction of 14-3-3 protein with phosphorylated Numbl-blocked dephosphorylation of Ser 304 . Taken together, these results indicate that the Numb family proteins may be intracellular targets for CaM-Ks, and they may also be regulated by phosphorylation-dependent interaction with 14-3-3 protein.

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Mechanism of the Generation of Autonomous Activity of Ca2+/Calmodulin-dependent Protein Kinase IV

Cited by 49 publications

References 48 publications

Ca2+/Calmodulin-Dependent Protein Kinase Kinase-α Regulates Skeletal Muscle Glucose Uptake Independent of AMP-Activated Protein Kinase and Akt Activation

Ca2+/Calmodulin-Dependent Protein Kinase Kinase-α Regulates Skeletal Muscle Glucose Uptake Independent of AMP-Activated Protein Kinase and Akt Activation

Pnck induces ligand-independent EGFR degradation by probable perturbation of the Hsp90 chaperone complex

Phosphorylation of Numb Family Proteins

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