Functional regulation of the protein phosphatase PPM1M by phosphorylation at multiple sites with Ser/Thr-Pro motifs.

The imbalance in the phosphorylation and the dephosphorylation of proteins leads to various diseases. Therefore, in vivo, the functions of protein kinases and protein phosphatases are strictly regulated. Mg2+/Mn2+-dependent protein phosphatase PPM1M has been implicated in immunity and cancer; however, the regulation mechanism remains unknown. In this study, we show that PPM1M is regulated in different ways by multiple phosphorylation. PPM1M has four Ser/Thr-Pro motifs (Ser27, Ser43, Ser60, and Thr254) that are recognized by proline-directed kinases, and Ser60 was found to be phosphorylated by cyclin-dependent kinase 5 (CDK5) in the cell. The phospho-mimetic mutation of Ser27 and Ser43 in the N-terminal domain suppresses the nuclear localization of PPM1M and promotes its accumulation in the cytoplasm. The phospho-mimetic mutation of Ser60 decreases PPM1M activity; conversely, the phospho-mimetic mutation of Thr254 increases PPM1M activity. These results suggest that the subcellular localization and phosphatase activity of PPM1M are regulated by protein kinases, including CDK5, via phosphorylation at multiple sites. Thus, PPM1M is differentially regulated by proline-directed kinases, including CDK5.

Subcellular distribution of bone morphogenetic protein 2-inducible kinase (BMP2K): Regulation by liquid-liquid phase separation and nucleocytoplasmic shuttling.

Bone morphogenetic protein 2 (BMP2)-inducible kinase (BMP2K) is induced by the cytokine BMP2, which is also implicated in the production of bone differentiation. In addition to regulating bone differentiation, BMP2K is implicated in a variety of cancers. Therefore, understanding the variables that determine where in the cell this kinase functions may help in understanding malignancies linked to BMP2K. However, the mechanisms regulating the subcellular localization of BMP2K are mainly unknown. By liquid-liquid phase separation (LLPS), BMP2K forms droplets in the cytoplasm, but how the droplets are regulated remains unclear. The reason why BMP2K localizes to the cytoplasm irrespective of having a nuclear localization signal (NLS) is also unknown. Here we show the element that controls BMP2K's LLPS and cytoplasmic localization. A glutamine-rich area is necessary for BMP2K phase separation, and droplet formation is controlled by hyperosmolarity. Cytoplasmic localization of BMP2K is managed by inhibition of NLS function through phosphorylation of Ser-1010 and by a newly found cytoplasmic localization region that antagonizes the NLS. These results will provide an important biochemical foundation for the advancement of BMP2K-related cell biology, structural biology, and pathophysiology.

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.

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.