Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways; how these enzymes are regulated is therefore of significant biological consequence. CK1s autophosphorylate their C-terminal non-catalytic tails, and eliminating these modifications increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and phosphoablating mutations increased Hhp1 and CK1ε activity towards substrates. Interestingly, substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. The presence or absence of tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, indicating that tails contribute to substrate specificity. Combining this mechanism with autophosphorylation of the T220 site in the catalytic domain, we propose a displacement specificity model to describe how autophosphorylation regulates substrate specificity for the CK1 family.

CaMK phosphatase (CaMKP/POPX2/PPM1F) inhibitors suppress the migration of human breast cancer MDA-MB-231 cells with loss of polarized morphology.

CaMK phosphatase (CaMKP/POPX2/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. Accumulating evidence suggests that CaMKP is involved in the pathogenesis of various diseases, including cancer. To clarify the relationship between CaMKP activity and human breast cancer cell motility, we examined the phosphatase activity of CaMKP in cell extracts. CaMKP activity assays of the immunoprecipitates prepared from the cell extract revealed that cells exhibiting higher motility had higher CaMKP activity, with no significant differences in the specific activity being observed. Two CaMKP-specific inhibitors, 1-amino-8-naphthol-4-sulfonic acid (ANS) and 1-amino-8-naphthol-2,4-disulfonic acid (ANDS), inhibited the migration of highly invasive MDA-MB-231 breast cancer cells without significant cytotoxicity, while an inactive analog, naphthionic acid, did not. Furthermore, the cells lost their elongated morphology and assumed a rounded shape following treatment with ANS, whereas they retained their elongated morphology following treatment with naphthionic acid. Consistent with these findings, ANS and ANDS significantly enhanced the phosphorylation level of CaMKI, a cellular substrate of CaMKP, while naphthionic acid did not. The present data suggest that CaMKP could be a novel therapeutic target for cancer metastasis.

CaM kinase phosphatase (CaMKP/PPM1F/POPX2) is specifically inactivated through gallate-mediated protein carbonylation.

CaMK phosphatase (CaMKP/PPM1F/POPX2) is a Mn2+-dependent, calyculin A/okadaic acid-insensitive Ser/Thr protein phosphatase that belongs to the PPM family. CaMKP is thought to be involved in regulation of not only various protein kinases, such as CaM kinases and p21-activated protein kinase, but also of cellular proteins regulated by phosphorylation. A large-scale screening of a chemical library identified gallic acid and some of its alkyl esters as novel CaMKP inhibitors highly specific to CaMKP. Surprisingly, they caused specific carbonylation of CaMKP, leading to its inactivation. Under the same conditions, no carbonylation nor inactivation was observed when PPM1A, which is affiliated with the same family as CaMKP, and λ-phosphatase were used. The carbonylation reaction was inhibited by SH compounds such as cysteamine in a dose-dependent manner with a concomitant decrease in CaMKP inhibition by ethyl gallate. The pyrogallol structure of gallate was necessary for the gallate-mediated carbonylation of CaMKP. Point mutations of CaMKP leading to impairment of phosphatase activity did not significantly affect the gallate-mediated carbonylation. Ethyl gallate resulted in almost complete inhibition of CaMKP under the conditions where the carbonylation level was nearly identical to that of CaMKP carbonylation via metal-catalyzed oxidation with ascorbic acid/FeSO4, which resulted in only a partial inhibition of CaMKP. The gallate-mediated carbonylation of CaMKP absolutely required divalent cations such as Mn2+, Cu2+, Co2+ and Fe2+, and was markedly enhanced by a phosphopeptide substrate. When MDA-MB-231 cells transiently expressing CaM kinase I, a CaMKP substrate, were treated by ethyl gallate, significant enhancement of phosphorylation of CaM kinase I was observed, suggesting that ethyl gallate can penetrate into cells to inactivate cellular CaMKP. All the presented data strongly support the hypothesis that CaMKP undergoes carbonylation of its specific amino acid residues by incubation with alkyl gallates and the divalent metal cations, leading to inactivation specific to CaMKP.

Biochemical characterization of four splice variants of mouse Ca2+/calmodulin-dependent protein kinase Iδ.

Ca2+/calmodulin (CaM)-dependent protein kinase Iδ (CaMKIδ) is a Ser/Thr kinase that plays pivotal roles in Ca2+ signalling. CaMKIδ is activated by Ca2+/CaM-binding and phosphorylation at Thr180 by CaMK kinase (CaMKK). In this study, we characterized four splice variants of mouse CaMKIδ (mCaMKIδs: a, b, c and d) found by in silico analysis. Recombinant mCaMKIδs expressed in Escherichia coli were phosphorylated by CaMKK; however, only mCaMKIδ-a and c showed protein kinase activities towards myelin basic protein in vitro, with mCaMKIδ-b and mCaMKIδ-d being inactive. Although mCaMKIδ-a and mCaMKIδ-c underwent autophosphorylation in vitro, only mCaMKIδ-c underwent autophosphorylation in 293T cells. Site-directed mutagenesis showed that the autophosphorylation site is Ser349, which is found in the C-terminal region of only variants c and b (Ser324). Furthermore, phosphorylation of these sites (Ser324 and Ser349) in mCaMKIδ-b and c was more efficiently catalyzed by cAMP-dependent protein kinase in vitro and in cellulo as compared to the autophosphorylation of mCaMKIδ-c. Thus, variants of mCaMKIδ possess distinct properties in terms of kinase activities, autophosphorylation and phosphorylation by another kinase, suggesting that they play physiologically different roles in murine cells.

Dual phosphorylation of protein phosphatase PPM1H promotes dephosphorylation of Smad1 in cellulo.

Protein phosphatase PPM1H is known to participate in various biological or pathophysiological mechanisms. However, little is known about the molecular mechanisms of its regulation. In this study, we investigated the protein kinases that directly phosphorylate PPM1H, identifying them as cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase I (CaMKI). In vitro and in silico analyses showed that the phosphorylation sites of PPM1H by PKA and CaMKI were Ser-123 and Ser-210, respectively. The phosphorylation state of PPM1H in cells exhibited the kinase activator- and inhibitor-dependent changes. In mouse neuroblastoma Neuro2a cells, phosphorylation of Ser-210 was much higher in the phospho-mimetic mutant (S123D) than in the non-phosphorylatable mutant (S123A) when they were treated with ionomycin. This suggests that a hierarchical phosphorylation, with initial phosphorylation of Ser-123 promoting subsequent phosphorylation of Ser-210, occurs in these neuron-like cells. Moreover, in cell-based assay a PPM1H(S123A/S210A) double mutant barely dephosphorylated Smad1, a transcription factor known as an endogenous substrate of PPM1H. These results suggest that cAMP and Ca2+/calmodulin regulate dephosphorylation of Smad1 through the dual phosphorylation of PPM1H at Ser-123 and Ser-210.

Autoactivation of C-terminally truncated Ca2+/calmodulin-dependent protein kinase (CaMK) Iδ via CaMK kinase-independent autophosphorylation.

Ca2+/calmodulin-dependent protein kinase I isoforms (CaMKIα, ß, γ, and δ) play important roles in Ca2+ signaling in eukaryotic cells by being activated by CaMK kinase (CaMKK) through phosphorylation at a Thr residue in the activation loop. However, we have recently found that, unlike rat CaMKIα (rCaMKIα), C-terminally truncated fragments of zebrafish and mouse CaMKIδ [zCaMKIδ(1-299) and mCaMKIδ(1-297)] produced by Escherichia coli exhibit almost full activity in the absence of CaMKK. To address the CaMKK-independent activation mechanism of CaMKIδ in E. coli cells, here we performed comparative analyses between recombinant zCaMKIδ(1-299) and rCaMKIα(1-294) in vitro. By using a kinase-dead mutant of zCaMKIδ(1-299) and λ phosphatase coexpression method, we elucidated that zCaMKIδ(1-299) was highly autophosphorylated and activated in E. coli during cell culture, but rCaMKIα(1-294) was not. The major autophosphorylation site leading to activation of the kinase was Ser296, determined using mass spectrometry analysis in conjunction with site-directed mutagenesis. Furthermore, mimicking phosphorylation at Ser296 in full-length zCaMKIδ resulted in additional activation of the kinase compared with CaMKI fully activated by CaMKK. Our results provide the first evidence that CaMKIδ is activated through CaMKK-independent phosphorylation at Ser296, which might be a clue to understand the physiological regulation of CaMKIδ isoform.

Facile preparation of highly active casein kinase 1 using Escherichia coli constitutively expressing lambda phosphatase.

Casein kinase 1 (CK1) is a widely expressed Ser/Thr kinase in eukaryotic organisms that is involved in various cellular processes (e.g., circadian rhythm and apoptosis). Therefore, preparing highly active CK1 and investigating its properties in vitro have important implications for understanding the biological roles of the kinase. However, recombinant CK1 undergoes autoinactivation via autophosphorylation in Escherichia coli cells and thus is undesirably prepared as a phosphorylated and inactivated kinase. To circumvent this problem, we established a protein expression system using E. coli strain BL21(DE3)pλPP in which λ protein phosphatase (λPPase) is constitutively expressed. Using this system, recombinant CK1 isoforms (α, δ and ε) were readily prepared as unphosphorylated forms. Furthermore, we found that CK1s prepared using BL21(DE3)pλPP showed markedly higher activity than those prepared by the conventional BL21(DE3). Finally, we demonstrated that the kinase activity of CK1δ from BL21(DE3)pλPP was higher than that prepared by a conventional method consisting of troublesome steps such as in vitro λPPase treatment. Thus, this simple method using BL21(DE3)pλPP is valuable for preparing highly active CK1s. It may also be applicable to other kinases that are difficult to prepare because of phosphorylation in E. coli cells.

High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli.

We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation.