Interdependent Phosphorylation within the Kinase Domain T-loop Regulates CHK2 Activity

Chk2 is a critical regulator of the cellular DNA damage repair response. Activation of Chk2 in response to IR-induced damage is initiated by phosphorylation of the Chk2 SQ/TQ cluster domain at Ser¹⁹, Ser³³, Ser³⁵, and Thr⁶⁸. This precedes autophosphorylation of Thr³⁸³/Thr³⁸⁷ in the T-loop region of the kinase domain an event that is a prerequisite for efficient kinase activity. We conducted an in-depth analysis of phosphorylation within the T-loop region (residues 366–406). We report four novel phosphorylation sites at Ser³⁷², Thr³⁷⁸, Thr³⁸⁹, and Tyr³⁹⁰. Substitution mutation Y390F was defective for kinase function. The substitution mutation T378A ablated the IR induction of kinase activity. Interestingly, the substitution mutation T389A demonstrated a 6-fold increase in kinase activity when compared with wild-type Chk2. In addition, phosphorylation at Thr³⁸⁹ was a prerequisite to phosphorylation at Thr³⁸⁷ but not at Thr³⁸³. Quantitative mass spectrometry analysis revealed IR-induced phosphorylation and subcellular distribution of Chk2 phosphorylated species. We observed IR-induced increase in phosphorylation at Ser³⁷⁹, Thr³⁸⁹, and Thr³⁸³/Thr³⁸⁹. Phosphorylation at Tyr³⁹⁰ was dramatically reduced following IR. Exposure to IR was also associated with changes in the ratio of chromatin/nuclear localization. IR-induced increase in chromatin localization was associated with phosphorylation at Thr³⁷², Thr³⁷⁹, Thr³⁸³, Thr³⁸⁹, Thr³⁸³/Thr³⁸⁷, and Thr³⁸³/Thr³⁸⁹. Chk2 hyper-phosphorylated species at Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹ and Thr³⁸³/Thr³⁸⁷/Thr³⁸⁹/Tyr³⁹⁰ relocalized from almost exclusively chromatin to predominately nuclear expression, suggesting a role for phosphorylation in regulation of chromatin targeting and egress. The differential impact of T-loop phosphorylation on Chk2 ubiquitylation suggests a co-dependence of these modifications. The results demonstrate that a complex interdependent network of phosphorylation events within the T-loop exchange region regulates dimerization/autophosphorylation, kinase activation, and chromatin targeting/egress of Chk2.     

The developmental stage and cell type dependent phosphorylation of eNOS in murine enteric mucosa and myenteric plexus

In order to clarify the developmental regulation of the eNOS activity in intestine by phosphorylation, we examined the immunohistochemical localizations of the eNOS phosphorylation sites at Ser¹¹⁷⁷, Ser¹¹⁶ and at Thr⁴⁹⁵ in cells of the mouse enteric mucosa and myenteric plexus at E13.5, E14.5, E16.5, E18.5, E20.5 and P3. In addition, in cells of the E16.5 stage the protein levels of eNOS and the phosphorylation sites of eNOS at Ser¹¹⁷⁷, Ser¹¹⁶ and at Thr⁴⁹⁵ were investigated by immunoblot. From E14.5 to P3, phosphorylation residues of eNOS at Ser¹¹⁷⁷ and at Ser¹¹⁶ were detected with different staining intensities in the enteric mucosa epithelium. In ganglion cells of the myenteric plexus Ser¹¹⁶ was identified at E18.5 to P3. The absence of phosphorylated Thr⁴⁹⁵ in cells of intestine during all developmental stages, was confirmed by immunoblot at E16.5. The immunoblot levels of eNOS and eNOS phosphorylated at Ser¹¹⁷⁷ and at Ser¹¹⁶ were comparable with the immunohistochemical results of E16.5 mouse intestine. It was concluded that development of epithelial cells of the enteric mucosa may be modulated by phosphorylation of eNOS at Ser¹¹⁷⁷ and at Ser¹¹⁶. The phosphorylation of eNOS in cells of the myenteric plexus is modulated at Ser¹¹⁶. These data suggest that there is a developmental stage and cell type dependent phosphorylation of eNOS.     

New Hierarchical Phosphorylation Pathway of the Translational Repressor eIF4E-binding Protein 1 (4E-BP1) in Ischemia-Reperfusion Stress

Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr⁶⁹-phosphorylated alone, Thr⁶⁹- and Thr³⁶/Thr⁴⁵-phosphorylated, all these plus Ser⁶⁴ phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr³⁶/Thr⁴⁵ phosphorylation alone was detected without Thr⁶⁹ phosphorylation, and neither was Ser⁶⁴ phosphorylation without Thr³⁶/Thr⁴⁵/Thr⁶⁹ phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr⁶⁹, Thr³⁶/Thr⁴⁵, and Ser⁶⁴ residues, with 4E-BP1 remaining phosphorylated at Thr⁶⁹ alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr³⁶/Thr⁴⁵ and Ser⁶⁴, in addition to Thr⁶⁹. Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr⁶⁹ is phosphorylated first followed by Thr³⁶/Thr⁴⁵ phosphorylation, and Ser⁶⁴ is phosphorylated last. Thr⁶⁹ phosphorylation alone allows binding to eIF4E, and subsequent Thr³⁶/Thr⁴⁵ phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis.     

Cross-talk between Rho-associated Kinase and Cyclic Nucleotide-dependent Kinase Signaling Pathways in the Regulation of Smooth Muscle Myosin Light Chain Phosphatase

Ca²⁺ sensitization of smooth muscle contraction depends upon the activities of protein kinases, including Rho-associated kinase, that phosphorylate the myosin phosphatase targeting subunit (MYPT1) at Thr⁶⁹⁷ and/or Thr⁸⁵⁵ (rat sequence numbering) to inhibit phosphatase activity and increase contractile force. Both Thr residues are preceded by the sequence RRS, and it has been suggested that phosphorylation at Ser⁶⁹⁶ prevents phosphorylation at Thr⁶⁹⁷. However, the effects of Ser⁸⁵⁴ and dual Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ phosphorylations on myosin phosphatase activity and contraction are unknown. We characterized a suite of MYPT1 proteins and phosphospecific antibodies for specificity toward monophosphorylation events (Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵), Ser phosphorylation events (Ser⁶⁹⁶/Ser⁸⁵⁴) and dual Ser/Thr phosphorylation events (Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵). Dual phosphorylation at Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ by cyclic nucleotide-dependent protein kinases had no effect on myosin phosphatase activity, whereas phosphorylation at Thr⁶⁹⁷ and Thr⁸⁵⁵ by Rho-associated kinase inhibited phosphatase activity and prevented phosphorylation by cAMP-dependent protein kinase at the neighboring Ser residues. Forskolin induced phosphorylation at Ser⁶⁹⁶, Thr⁶⁹⁷, Ser⁸⁵⁴, and Thr⁸⁵⁵ in rat caudal artery, whereas U46619 induced Thr⁶⁹⁷ and Thr⁸⁵⁵ phosphorylation and prevented the Ser phosphorylation induced by forskolin. Furthermore, pretreatment with forskolin prevented U46619-induced Thr phosphorylations. We conclude that cross-talk between cyclic nucleotide and RhoA signaling pathways dictates the phosphorylation status of the Ser⁶⁹⁶–Thr⁶⁹⁷ and Ser⁸⁵⁴–Thr⁸⁵⁵ inhibitory regions of MYPT1 in situ, thereby regulating the activity of myosin phosphatase and contraction.     

Identification of phosphorylation sites in the COOH‐terminal tail of the μ‐opioid receptor

Phosphorylation is considered a key event in the signalling and regulation of the μ opioid receptor (MOPr). Here, we used mass spectroscopy to determine the phosphorylation status of the C‐terminal tail of the rat MOPr expressed in human embryonic kidney 293 (HEK‐293) cells. Under basal conditions, MOPr is phosphorylated on Ser³⁶³ and Thr³⁷⁰, while in the presence of morphine or [D‐Ala2, NMe‐Phe4, Gly‐ol5]‐enkephalin (DAMGO), the COOH terminus is phosphorylated at three additional residues, Ser³⁵⁶, Thr³⁵⁷ and Ser³⁷⁵. Using N‐terminal glutathione S transferase (GST) fusion proteins of the cytoplasmic, C‐terminal tail of MOPr and point mutations of the same, we show that, in vitro, purified G protein‐coupled receptor kinase 2 (GRK2) phosphorylates Ser³⁷⁵, protein kinase C (PKC) phosphorylates Ser³⁶³, while CaMKII phosphorylates Thr³⁷⁰. Phosphorylation of the GST fusion protein of the C‐terminal tail of MOPr enhanced its ability to bind arrestin‐2 and ‐3. Hence, our study identifies both the basal and agonist‐stimulated phospho‐acceptor sites in the C‐terminal tail of MOPr, and suggests that the receptor is subject to phosphorylation and hence regulation by multiple protein kinases.     

Methylglyoxal Activates the Target of Rapamycin Complex 2-Protein Kinase C Signaling Pathway in Saccharomyces cerevisiae

Methylglyoxal is a typical 2-oxoaldehyde derived from glycolysis. We show here that methylglyoxal activates the Pkc1-Mpk1 mitogen-activated protein (MAP) kinase cascade in a target of rapamycin complex 2 (TORC2)-dependent manner in the budding yeast Saccharomyces cerevisiae. We demonstrate that TORC2 phosphorylates Pkc1 at Thr¹¹²⁵ and Ser¹¹⁴³. Methylglyoxal enhanced the phosphorylation of Pkc1 at Ser¹¹⁴³, which transmitted the signal to the downstream Mpk1 MAP kinase cascade. We found that the phosphorylation status of Pkc1^T1125 affected the phosphorylation of Pkc1 at Ser¹¹⁴³, in addition to its protein levels. Methylglyoxal activated mammalian TORC2 signaling, which, in turn, phosphorylated Akt at Ser⁴⁷³. Our results suggest that methylglyoxal is a conserved initiator of TORC2 signaling among eukaryotes.     

Phosphorylation of neuronal nitric oxide synthase at Ser in the dentate gyrus of rat brain after transient forebrain ischemia

We previously demonstrated that calmodulin-dependent protein kinase IIα (CaM-KIIα) phosphorylates nNOS at Ser⁸⁴⁷ in the hippocampus after forebrain ischemia; this phosphorylation attenuates NOS activity and might contribute to resistance to post-ischemic damage. We also revealed that cyclic AMP-dependent protein kinase (PKA) could phosphorylate nNOS at Ser¹⁴¹²in vitro. In this study, we focused on chronological and topographical changes in the phosphorylation of nNOS at Ser¹⁴¹² after rat forebrain ischemia. The hippocampus and adjacent cortex were collected at different times, up to 24 h, after 15 min of forebrain ischemia. NOS was partially purified from crude samples using ADP agarose gel. Neuronal NOS, phosphorylated (p)-nNOS at Ser¹⁴¹², PKA, and p-PKA at Thr¹⁹⁷ were studied in the rat hippocampus and cortex using Western blot analysis and immunohistochemistry. Western blot analysis revealed that p-nNOS at Ser¹⁴¹² significantly increased between 1 and 6 h after reperfusion in the hippocampus, but not in the cortex. PKA was cosedimented with nNOS by ADP agarose gel. Immunohistochemistry revealed that phosphorylation of nNOS at Ser¹⁴¹² and PKA at Thr¹⁹⁷ occurred in the subgranular layer of the dentate gyrus. Forebrain ischemia might thereby induce temporary activation of PKA at Thr¹⁹⁷, which then phosphorylates nNOS at Ser¹⁴¹² in the subgranular layer of the dentate gyrus.     

Phospholamban phosphorylation in ischemia-reperfused heart. Effect of pacing during ischemia and response to a β-adrenergic challenge

The status of phospholamban (PLB) phosphorylation in the ischemia-reperfused hearts remains controversial. Although a decrease in the phosphorylation of both PLB residues (Ser¹⁶, PKA site, and Thr¹⁷, CaMKII site) was previously reported, experiments from our laboratory failed to detect this decrease. In an attempt to elucidate the cause for this discrepancy, experiments were performed in Langendorff-perfused rat hearts with two main goals: (1) To determine whether keeping pacing during ischemia, a protocol followed in other ischemia-reperfusion models, decreases the phosphorylation of PLB residues, below pre-ischemic values; (2) To investigate whether a maximal -adrenergic challenge allows to detect a decrease in the ability of PLB to be phosphorylated in ischemia-reperfused hearts. Hearts were submitted to a global ischemia/reperfusion protocol (20/30 min) with (P) or without (NP) pacing during ischemia, and phosphorylation of PLB residues was assessed by immunodetection. The recovery of contractility upon reperfusion was lower in P vs. NP hearts. Ser¹⁶ of PLB, was phosphorylated at the end of ischemia in NP hearts. This increase appeared earlier in P hearts and was significantly diminished by catecholamine depletion and -blockade. Thr¹⁷ site was phosphorylated at the beginning of ischemia and the onset of reperfusion. The ischemia-induced phosphorylation of Thr¹⁷ was higher and more sustained in P vs. NP hearts, and inhibited by the calcium channel blocker, nifedipine, whereas the reperfusion-induced increase in Thr¹⁷ phosphorylation was similar in P and NP hearts and was significantly diminished by the Na⁺/Ca²⁺ exchanger inhibitor KB-R7943. Phosphorylation of PLB residues did not decrease below basal levels at any time during ischemia and reperfusion. However, the phosphorylation, inotropic and lusitropic response to -adrenergic stimulation was significantly decreased both in P and NP hearts.     

Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis

The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser²⁰⁵⁶, Thr²⁶⁴⁷ and Thr²⁶⁰⁹ in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser³²⁰⁵ and Thr³⁹⁵⁰ in mitosis. Phosphorylation of Thr³⁹⁵⁰ is DNA-PK-dependent, whereas phosphorylation of Ser³²⁰⁵ requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser³²⁰⁵ in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser³²⁰⁵ in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr⁶⁸ in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs’ role in mitosis may be mechanistically distinct from its well-established role in NHEJ.     

Tobacco Calcium-dependent Protein Kinases Are Differentially Phosphorylated in Vivo as Part of a Kinase Cascade That Regulates Stress Response

In vivo phosphorylation sites of the tobacco calcium-dependent protein kinases NtCDPK2 and NtCDPK3 were determined in response to biotic or abiotic stress. Stress-inducible phosphorylation was exclusively located in the variable N termini, where both kinases were phosphorylated differentially despite 91% overall sequence identity. In NtCDPK2, serine 40 and threonine 65 were phosphorylated within 2 min after stress. Whereas Thr⁶⁵ is subjected to intra-molecular in vivo autophosphorylation, Ser⁴⁰ represents a target for a regulatory upstream protein kinase, and correct NtCDPK2 membrane localization is required for Ser⁴⁰ phosphorylation. NtCDPK3 is phosphorylated at least at two sites in the N terminus by upstream kinase(s) upon stress stimulus, first at Ser⁵⁴, a site not present in NtCDPK2, and also at a second undetermined site not identical to Ser⁴⁰. Domain swap experiments established that differential phosphorylation of both kinases is exclusively determined by the respective N termini. A cell death-inducing response was only observed upon expression of a truncated variant lacking the junction and calcium-binding domain of NtCDPK2 (VK2). This response required protein kinase activity and was reduced when subcellular membrane localization was disturbed by a mutation in the myristoylation and palmitoylation site. Our data indicate that CDPKs are integrated in stress-dependent protein kinase signaling cascades, and regulation of CDPK function in response to in vivo stimulation is dependent on its membrane localization.     

Phosphorylation of phospholamban in ischemia-reperfusion injury: Functional role of Thr17 residue

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser¹⁶ by PKA and/or at Thr¹⁷ by CaMKII increases the affinity of the SR Ca²⁺ pump for Ca²⁺. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr¹⁷ PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser¹⁶ and Thr¹⁷ residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr¹⁷ phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr¹⁷ phosphorylation was dependent on Ca²⁺ entry to the cardiac cell. This Ca²⁺ influx would mainly occur by the coupled activation of the Na⁺/H⁺ exchanger and the Na⁺/Ca²⁺ exchanger working in the reverse mode, since phosphorylation of Thr¹⁷ was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca²⁺ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr¹⁷ phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr¹⁷ of PLB upon reflow, may favor the full recovery of relaxation after ischemia. (Mol Cell Biochem 263: 131–136, 2004)     

Simian Virus 40 Large T Antigen Is Phosphorylated at Multiple Sites Clustered in Two Separate Regions

The phosphorylation sites of simian virus 40 large T antigen were determined within the primary structure of the molecule. Exhaustive digestion of ³²P-labeled large T antigen with trypsin generated six major phosphopeptides which could be separated in a newly developed isobutyric acid-containing chromatography system. By partial tryptic digestion, large T antigen was cleaved into an amino-terminal fragment of 17,000 daltons and overlapping fragments from the carboxy-terminal region ranging in size between 71,000 and 13,000 daltons. The location of the phosphopeptides was then determined by fingerprint analyses of individual fragments. Their physical properties were analyzed by sizing on polyacrylamide gels and by sequential digestion and peptide mapping; their amino acid composition was determined by differential labeling with various amino acids. The amino-terminal 17,000-dalton fragment gave rise to only one phosphopeptide (phosphopeptide 3) that contained half of the phosphate label incorporated into large T antigen. It contained phosphoserine and phosphothreonine sites, all of which were clustered within a small segment between Cys₁₀₅ and Lys₁₂₇. This segment contained five serines and two threonines. Among these, Ser₁₀₆, Ser₁₂₃, and Thr₁₂₄ were identified as phosphorylated residues; in addition, either one or both of Ser₁₁₁ and Ser₁₁₂ were phosphorylated. The neighboring residues, Ser₁₂₃ and Thr₁₂₄, were found in three different phosphorylation states in that either Ser₁₂₃ or Thr₁₂₄ or both were phosphorylated. Phosphopeptides 1, 2, 4, 5, and 6 were all derived from a single fragment extending 26,000 daltons upstream from the carboxy terminus of large T antigen. Phosphopeptide 6 was identical with the previously determined phosphothreonine peptide phosphorylated at Thr₇₀₁. Phosphopeptides 1, 2, 4, and 5 contained only serine-bound phosphate. Phosphopeptides 1, 2, and 4 represented overlapping peptides, all of which were phosphorylated at Ser₆₃₉ located next to a cluster of six acidic residues. In phosphopeptide 5, a large peptide ranging from Asn₆₅₃ to Arg₆₉₁, at least two of seven serines were phosphorylated. Thus, large T antigen contains at least eight phosphorylation sites. Their clustering within two separate regions might correlate with structural and functional domains of this protein.     

Regulation of mTOR Complex 1 (mTORC1) by Raptor Ser863 and Multisite Phosphorylation

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser⁶⁹⁶/Thr⁷⁰⁶ and cluster 2, Ser⁸⁵⁵/Ser⁸⁵⁹/Ser⁸⁶³/Ser⁸⁷⁷) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser⁸⁶³ phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser⁸⁶³ via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser⁸⁶³ phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser⁸⁶³ as well as on the five other identified sites (e.g. Ser⁸⁵⁹, Ser⁸⁵⁵, Ser⁸⁷⁷, Ser⁶⁹⁶, and Thr⁷⁰⁶). Strikingly, raptor Ser⁸⁶³ phosphorylation is absolutely required for raptor Ser⁸⁵⁹ and Ser⁸⁵⁵ phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser⁸⁶³ phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser⁸⁵⁹ and Ser⁸⁵⁵). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.     

A Novel Regulatory Locus of Phosphorylation in the C Terminus of the Potassium Chloride Cotransporter KCC2 That Interferes with N-Ethylmaleimide or Staurosporine-mediated Activation

The neuron-specific cation chloride cotransporter KCC2 plays a crucial role in hyperpolarizing synaptic inhibition. Transporter dysfunction is associated with various neurological disorders, raising interest in regulatory mechanisms. Phosphorylation has been identified as a key regulatory process. Here, we retrieved experimentally observed phosphorylation sites of KCC2 from public databases and report on the systematic analysis of six phosphorylated serines, Ser²⁵, Ser²⁶, Ser⁹³⁷, Ser¹⁰²², Ser¹⁰²⁵, and Ser¹⁰²⁶. Alanine or aspartate substitutions of these residues were analyzed in HEK-293 cells. All mutants were expressed in a pattern similar to wild-type KCC2 (KCC2^WT). Tl⁺ flux measurements demonstrated unchanged transport activity for Ser²⁵, Ser²⁶, Ser¹⁰²², Ser¹⁰²⁵, and Ser¹⁰²⁶ mutants. In contrast, KCC2^S937D, mimicking phosphorylation, resulted in a significant up-regulation of transport activity. Aspartate substitution of Thr⁹³⁴, a neighboring putative phosphorylation site, resulted in a comparable increase in KCC2 transport activity. Both KCC2^T934D and KCC2^S937D mutants were inhibited by the kinase inhibitor staurosporine and by N-ethylmaleimide, whereas KCC2^WT, KCC2^T934A, and KCC2^S937A were activated. The inverse staurosporine effect on aspartate versus alanine substitutions reveals a cross-talk between different phosphorylation sites of KCC2. Immunoblot and cell surface labeling experiments detected no alterations in total abundance or surface expression of KCC2^T934D and KCC2^S937D compared with KCC2^WT. These data reveal kinetic regulation of transport activity by these residues. In summary, our data identify a novel key regulatory phosphorylation site of KCC2 and a functional interaction between different conformation-changing post-translational modifications. The action of pharmacological agents aimed to modulate KCC2 activity for therapeutic benefit might therefore be highly context-specific.     

M Phase-Specific Phosphorylation of Histone H1.5 at Threonine 10 by GSK-3

H1 histones are progressively phosphorylated during the cell cycle. The number of phosphorylated sites is zero to three in late S phase and increases to five or six in late G2 phase and M phase. It is assumed that this phosphorylation modulates chromatin condensation and decondensation, but its specific role remains unclear. Recently, it was shown that the somatic H1 histone subtype H1.5 becomes pentaphosphorylated during mitosis, with phosphorylated threonine 10 being the last site to be phosphorylated. We have generated an antiserum specific for human H1.5 phosphorylated at threonine 10. Immunofluorescence labeling of HeLa cells with this antiserum revealed that the phosphorylation at this site appears in prometaphase and disappears in telophase, and that this hyperphosphorylated form of H1.5 is mainly chromatin-bound in metaphase when chromatin condensation is maximal. In search of the kinase responsible for the phosphorylation at this site, we found that threonine 10 of H1.5 can be phosphorylated by glycogen synthase kinase-3 in vitro, but not by cyclin-dependent kinase 1/cyclin B and cyclin-dependent kinase 5/p35, respectively. Furthermore, addition of specific glycogen synthase kinase-3 inhibitors led to a reduction in phosphorylation at this site both in vivo and in vitro.     

Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue

The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1–3 kinases are specifically activated by two phosphorylation events on residues Thr³⁰⁸ and Ser⁴⁷³ upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser⁴⁷³ and Thr³⁰⁸ phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser⁴⁷³ and Thr³⁰⁸ phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser⁴⁷³-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.