Mechanism Of Autophosphorylation Research Articles

Phosphorylation-Competent Metastable State of Escherichia coli Toxin HipA.

Phosphorylation is a key post-translational modification that alters the functional state of many proteins. The Escherichia coli toxin HipA, which phosphorylates glutamyl-tRNA synthetase and triggers bacterial persistence under stress, becomes inactivated upon autophosphorylation of Ser150. Interestingly, Ser150 is phosphorylation-incompetent in the crystal structure of HipA since it is deeply buried ("in-state"), although in the phosphorylated state it is solvent exposed ("out-state"). To be phosphorylated, a minor population of HipA must exist in the phosphorylation-competent "out-state" (solvent-exposed Ser150), not detected in the crystal structure of unphosphorylated HipA. Here we report a molten-globule-like intermediate of HipA at low urea (∼4 kcal/mol unstable than natively folded HipA). The intermediate is aggregation-prone, consistent with a solvent exposed Ser150 and its two flanking hydrophobic neighbors (Val/Ile) in the "out-state". Molecular dynamics simulations showed the HipA "in-out" pathway to contain multiple free energy minima with an increasing degree of Ser150 solvent exposure with the free energy difference between the "in-state" and the metastable exposed state(s) to be ∼2-2.5 kcal/mol, with unique sets of hydrogen bonds and salt bridges associated with the metastable loop conformations. Together, the data clearly identify the existence of a phosphorylation-competent metastable state of HipA. Our results not only suggest a mechanism of HipA autophosphorylation but also add to a number of recent reports on unrelated protein systems where the common proposed mechanism for phosphorylation of buried residues is their transient exposure even without phosphorylation.

Crystal structure of the Escherichia coli CusS kinase core

The CusS histidine kinase is a member of Escherichia coli two-component signal transduction system, engaged in a response to copper ions excess in the cell periplasm. The periplasmic sensor domain of CusS binds the free copper ions and the CusS kinase core phosphorylates the cognate CusR which regulates transcription of the efflux pomp CusCBA. A small amount of copper ions is indispensable for the aerobic cell metabolism. Nonetheless, its excess in the cytoplasm generates damaging and reactive hydroxyl radicals. For that reason, understanding the bacterial copper sensing mechanisms can contribute to reducing bacterial copper-resistance and developing bactericidal copper-based materials. The crystal structure of the CusS kinase core was solved at the resolution of 1.4 Å. The cytoplasmic catalytic core domains formed a homodimer. Based on the obtained structure, the intramolecular and intermolecular interactions crucial for the mechanism of CusS autophosphorylation were described.

Regulation of RAS function by active site autophosphorylation

A unifying feature of the RAS superfamily is a functionally conserved cycle that small GTPases use to transition between active and inactive states. Here, we demonstrate that active site autophosphorylation of the oncogenic mutant K-RASA59T is an irreversible and intrinsic regulatory mechanism that alters nucleotide exchange, effector interaction, and downstream signaling. We used x-ray crystallography, enzymology, molecular dynamics and cell assays to explore the mechanism of autophosphorylation and its impact on RAS function. Our crystal structures of H-RASA59T support a zero-order and intramolecular mechanism of autophosphorylation that involves direct attack of threonine 59 on GTP. Further, we find that post-phosphorylation, intrinsic nucleotide exchange is enhanced and favored over GEF mediated nucleotide exchange. Our crystal structures of H-RASA59E and K-RASA59E support this difference in biochemical activity. The impact of active site reorganization extends beyond stabilization of the nucleotide bound state to RAS effector interactions and utilization. Using effector binding assays, as well as classic assays of K-RAS cell transformation, our data demonstrate that K-RASA59T and K-RASA59E do not depend on RAF kinases for cell transformation. Finally, we used structural analysis and molecular dynamics to show that other small GTPases with a conserved threonine or serine at residue 59 likely undergo autophosphorylation. Many of these small GTPases have similar biochemical features to K-RASA59T, including overall stabilization of the GTP versus GDP bound state. We speculate that autophosphorylation is an unexplored means of regulation for these atypical small GTPases.

Autophosphorylation of MAP kinase disables the MAPK pathway in apoptotic Xenopus eggs

Mitogen-activated protein kinases (MAPKs) are involved in the regulation of various cellular processes, including cell survival and apoptosis. Here, we report that Xenopus p42 MAPK becomes phosphorylated in apoptotic eggs, however this modification does not activate the enzyme. Using phosphorylation residue-specific antibodies, we demonstrate that this modification occurs on the Tyr residue in the MAPK activation segment, pinpointing the autophosphorylation mechanism. Notably, MAPK phosphorylation in apoptotic Xenopus eggs coincides with prominent intracellular acidification accompanying apoptosis in these cells. Furthermore, autophosphorylation of recombinant Xenopus MAPK is stimulated and phosphorylation of a protein substrate is inhibited under low pH conditions. Thus, acidic intracellular conditions inactivate MAPK and effectively disable the MAPK-mediated survival pathway in the apoptotic eggs. Given that cell acidification is a rather common feature of apoptosis, we hypothesize that stimulation of MAPK autophosphorylation and shutdown of the MAPK pathway may represent universal traits of apoptotic cell death.

Autophosphorylation Mechanism of the Ser/Thr Kinase Stk1 From Staphylococcus aureus.

The eukaryotic-like Ser/Thr kinase Stk1 is crucial for virulence, cell wall biosynthesis, and drug susceptibility in methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA). Importantly, MRSA lacking Stk1 become sensitive to β-lactam antibiotics, implying that Stk1 could be an alternative target for combination therapy. However, the autophosphorylation mechanism of Stk1 remains elusive. Using a phosphoproteomic study, we identified six in vivo phosphorylated activation loop residues (Ser159, Thr161, Ser162, Thr164, Thr166, and Thr172) of Stk1, which are also phosphorylated in vitro. We further showed that cis autophosphorylation of Thr172 in the GT/S motif is essential for self-activation and kinase activity of Stk1 kinase domain (Stk1-KD), whereas the trans autophosphorylation of other activation loop serines/threonines are required for the optimal kinase activity of Stk1-KD. Moreover, substitution of the activation loop serines/threonines impaired in vivo autophosphorylation activity of kinase variants, while T172A and T172D variants were unable to autophosphorylate in the cellular content, underlining the essential role of Thr172 for Stk1 activity in vivo. This study provides insights into molecular basis for regulation of Stk1 activity from S. aureus.

EDHSS206, an Exquisitely Selective Inhibitor of TAK1, Targets Pro Survival TNFFFDependent Signaling, Inducing Apoptosis in Rheumatoid Arthritis and Breast Cancer Models

Tumor necrosis factor alpha (TNFα) administration has shown limited therapeutic utility in cancer and inflammatory disorders due to dose‐limiting side effects. We propose an alternative approach to targeting TNFα‐mediated diseases by selectively inducing the TNFα apoptotic response in TNFα‐rich microenvironments. TAK1 acts as a key mediator between survival and cell death in TNFα signaling. We report a novel potent and exquisitely selective TAK1 inhibitor (EDHS‐206, IC50 9.5nM) that induces apoptosis in a TNFα‐dependent manner in models of metastatic breast cancer and rheumatoid arthritis. Co‐crystallization studies demonstrated that EDHS‐206 inhibits the kinase in a DFG‐in conformation. Kinetic analysis of EDHS‐206‐TAK1 interactions revealed a substrate‐like intermolecular autophosphorylation mechanism for TAK1 activation, during which EDHS‐206 delays the proceedings of the rate‐limiting step. EDHS‐206 represents an attractive starting point for the development of a novel generation of inhibitors that greatly sensitize cells to TNFα‐induced cell death, potentially broadening the therapeutic efficacy of TNFα.

Molecular and biochemical characterization of a Vigna mungo MAP kinase associated with Mungbean Yellow Mosaic India Virus infection and deciphering its role in restricting the virus multiplication

Yellow Mosaic Disease caused by the begomovirus Mungbean Yellow Mosaic India Virus (MYMIV) severely affects many economically important legumes. Recent investigations in Vigna mungo – MYMIV incompatible interaction identified a MAPK homolog in the defense signaling pathway. An important branch of immunity involves phosphorylation by evolutionary conserved Mitogen-activated protein kinases (MAPK) that transduce signals of pathogen invasion to downstream molecules leading to diverse immune responses. However, most of the knowledge of MAPKs is derived from model crops, and functions of these versatile kinases are little explored in legumes. Here we report characterization of a MAP kinase (VmMAPK1), which was induced upon MYMIV-inoculation in resistant V. mungo. Phylogenetic analysis revealed that VmMAPK1 is closely related to other plant-stress-responsive MAPKs. Both mRNA and protein of VmMAPK1 were accumulated upon MYMIV infection. The VmMAPK1 protein localized in the nucleus as well as cytoplasm and possessed phosphorylation activity in vitro. A detailed biochemical characterization of purified recombinant VmMAPK1 demonstrated an intramolecular mechanism of autophosphorylation and self-catalyzed phosphate incorporation on both threonine and tyrosine residues. The Vmax and Km values of recombinant VmMAPK1 for ATP were 6.292nmol/mg/min and 0.7978μM, respectively. Furthermore, the ability of VmMAPK1 to restrict MYMIV multiplication was validated by its ectopic expression in transgenic tobacco. Importantly, overexpression of VmMAPK1 resulted in the considerable upregulation of defense-responsive marker PR genes. Thus, the present data suggests the critical role of VmMAPK1 in suppressing MYMIV multiplication presumably through SA-mediated signaling pathway and inducing PR genes establishing the significant implications in understanding MAP kinase gene function during Vigna-MYMIV interaction; and hence paves the way for introgression of resistance in leguminous crops susceptible to MYMIV.

A mathematical model of Aurora B activity in prophase and metaphase

Aurora B kinase is a protein that controls several processes in mitosis when it is found associated with INCENP, Survivin and Borealin in a complex known as the Chromosomal Passenger Complex. Aurora B in complex with INCENP is phosphorylated on three sites, resulting in the full activation of Aurora B. In prophase and metaphase, Aurora B is activated at centromeres, the region of chromatin linking sister chromatids, due to an autophosphorylation mechanism, and it has been hypothesised that Aurora B is activated throughout the cytoplasm due to its concentration at centromeres. In this article, we first develop a time-dependent model of Aurora B activation that does not incorporate spatial variation. This model is used to demonstrate the various qualitative behaviours that the activation of Aurora B is capable of displaying for different model parameters. Next, we develop a spatio-temporal model of Aurora B activation that includes diffusion of soluble Aurora B and binding of Aurora B to immobile centromeric binding sites. This model describes the activation of Aurora B throughout the cytoplasm due to its concentration-dependent activation at centromeres. The models demonstrate the effects that a soluble phosphatase concentration, multisite phosphorylation and diffusion have on the activation of Aurora B.

Snapshots of P-Type ATPases - From Crystal Structures to Single-Molecule Studies

P-type ATPases maintain essential ion gradients and asymmetric distributions of lipids across cell membranes. They encompass well-known ion pumps such as the Na+,K+-ATPase and Ca2+-ATPases. Much knowledge has been obtained on general principles of P-type ATPase structure, function and inhibition, including a vast database of P-type ATPase structures, mutational studies and transport kinetics studies. Our studies have revealed large conformational changes associated with the Post-Albers transport cycle of e.g. sarcoendoplamic reticulum Ca2+-ATPase and pig kidney Na,K-ATPase, where the enzyme alternates between inward-oriented E1 forms and outward-oriented E2 forms. We also seek to contribute with new structures representing missing functional states or inhibited forms, regulatory mechanisms and complexes, and structures of P-type ATPases which have remained so far uncharacterized. Overall, the cycle is driven in a forward direction by ATP hydrolysis through a two-step mechanism of autophosphorylation (E1 to E1P) and autodephosphorylation (E2P to E2) of the enzyme. Critical transitions take place when the orientation shifts sides (E1P to E2P and E2 to E1). However, most information on the cycle has so far been derived from bulk measurements, and insights into the actual coupling and kinetics at a single-molecule level has been missing. We have established a P-type ATPase model for single-molecule studies based on the Listeria Ca2+-ATPase 1 (LMCA1). Laursen M et al. 2015. Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex. PNAS 112:1755 Nyblom M et al. 2013. Crystal Structure of Na+, K+-ATPase in the Na+-Bound State. Science 342:123 Winther AM et al. 2013. The Sarcolipin-bound Calcium Pump Stabilizes Calcium Sites exposed to the Cytoplasm. Nature 495:265 Faxen K et al. 2011. Characterisation of a Listeria monocytogenes calcium pump - a SERCA type ATPase with only one Ca2+-binding site. J Biol Chem 286:1609

Regulation of PKA activity by an autophosphorylation mechanism in Saccharomyces cerevisiae.

PKA (cAMP-dependent protein kinase) activity, as well as that of other AGC members, is regulated by multiple phosphorylations of its catalytic subunits. In Saccharomyces cerevisiae, the PKA regulatory subunit is encoded by the gene BCY1, and the catalytic subunits are encoded by three genes: TPK1, TPK2 and TPK3. Previously, we have reported that, following cAMP/PKA pathway activation, Tpk1 increases its phosphorylation status. Now, invivo genetic and invitro experiments indicate an autophosphorylation mechanism for Tpk1. Using array peptides derived from Tpk1, we identified Ser179 as a target residue. Tpk1 is phosphorylated on Ser179 invivo during glucose stimulus. Reduction of the activation loop Thr241 phosphorylation increases Ser179 autophosphorylation. To evaluate the role of phosphorylation on Ser179, we made strains expressing tpk1S179A or tpk1S179D as the sole PKA kinase source. Our results suggest that Ser179 phosphorylation increases the reactivity towards the substrate without affecting the formation of the holoenzyme. Phenotypic readout analysis showed that Ser179 phosphorylation increases invivo PKA activity, reducing cell survival, stress and lifespan. Ser179 phosphorylation increases Tpk1 cytoplasmic accumulation in glucose-grown cells. These results describe for the first time that an autophosphorylation mechanism on Tpk1 controls PKA activity in response to glucose availability.

Mechanism of autophosphorylation of mycobacterial PknB explored by molecular dynamics simulations.

Mycobacterial Ser/Thr kinase, PknB, is essential for the growth of the pathogen. Unphosphorylated PknB is catalytically inactive, and its activation requires autophosphorylation of Thr residues on the activation loop. Autophosphorylation can in principle take place via two distinct mechanisms. Intermolecular trans autophosphorylation involves dimerization and phosphorylation of the activation loop of one chain in the catalytic pocket of the other chain. On the other hand, intramolecular cis autophosphorylation involves phosphorylation of the activation loop of the kinases in its own catalytic pocket within a monomer. On the basis of the crystal structure of PknB in the front-to-front dimeric form, it is currently believed that activation of PknB involves trans autophosphorylation. However, because of the lack of coordinates of the activation loop in the crystal structures, atomic details of the conformational changes associated with activation are yet to be deciphered. Therefore, to understand the conformational transitions associated with activation via autophosphorylation, a series of explicit solvent molecular dynamics simulations with a duration of 1 μs have been performed on each of the phosphorylated and nonphosphorylated forms of the PknB catalytic domain in monomeric and dimeric states. Simulations on phosphorylated PknB revealed a differential network of crucial electrostatic and hydrophobic residues that stabilize the phosphorylated form in the active conformation. Interestingly, in our simulations on nonphosphorylated monomers, the activation loop was observed to fold into its own active site, thereby opening the novel possibility of activation through intramolecular cis autophosphorylation. Thus, our simulations suggest that autophosphorylation of PknB might also involve cis initiation followed by trans amplification as reported for other eukaryotic kinases based on recent reaction kinetics studies.

Molecular mechanism of Aurora A kinase autophosphorylation and its allosteric activation by TPX2.

We elucidate the molecular mechanisms of two distinct activation strategies (autophosphorylation and TPX2-mediated activation) in human Aurora A kinase. Classic allosteric activation is in play where either activation loop phosphorylation or TPX2 binding to a conserved hydrophobic groove shifts the equilibrium far towards the active conformation. We resolve the controversy about the mechanism of autophosphorylation by demonstrating intermolecular autophosphorylation in a long-lived dimer by combining X-ray crystallography with functional assays. We then address the allosteric activation by TPX2 through activity assays and the crystal structure of a domain-swapped dimer of dephosphorylated Aurora A and TPX2(1-25). While autophosphorylation is the key regulatory mechanism in the centrosomes in the early stages of mitosis, allosteric activation by TPX2 of dephosphorylated Aurora A could be at play in the spindle microtubules. The mechanistic insights into autophosphorylation and allosteric activation by TPX2 binding proposed here, may have implications for understanding regulation of other protein kinases.DOI: http://dx.doi.org/10.7554/eLife.02667.001.

Structural basis of Rad53 kinase activation by dimerization and activation segment exchange

The protein kinase Rad53 is a key regulator of the DNA damage checkpoint in budding yeast. Its human ortholog, CHEK2, is mutated in familial breast cancer and mediates apoptosis in response to genotoxic stress. Autophosphorylation of Rad53 at residue Thr354 located in the kinase activation segment is essential for Rad53 activation. In this study, we assessed the requirement of kinase domain dimerization and the exchange of its activation segment during the Rad53 activation process. We solved the crystal structure of Rad53 in its dimeric form and found that disruption of the observed head-to-tail, face-to-face dimer structure decreased Rad53 autophosphorylation on Thr354 in vitro and impaired Rad53 function in vivo. Moreover, we provide critical functional evidence that Rad53 trans-autophosphorylation may involve the interkinase domain exchange of helix αEF via an invariant salt bridge. These findings suggest a mechanism of autophosphorylation that may be broadly applicable to other protein kinases.

Visualizing autophosphorylation in histidine kinases

Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis- and trans-phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality.

Multiple layers of regulation influence cell integrity control by the PKC ortholog Pck2 in fission yeast.

The fission yeast protein kinase C (PKC) ortholog Pck2 controls cell wall synthesis and is a major upstream activator of the cell integrity pathway (CIP) and its core component, the MAP kinase Pmk1 (also known as Spm1), in response to environmental stimuli. We show that in vivo phosphorylation of Pck2 at the conserved T842 activation loop during growth and in response to different stresses is mediated by the phosphoinositide-dependent kinase (PDK) ortholog Ksg1 and an autophosphorylation mechanism. However, T842 phosphorylation is not essential for Pmk1 activation, and putative phosphorylation at T846 might play an additional role in Pck2 catalytic activation and downstream signaling. These events, together with turn motif autophosphorylation at T984 and binding to small GTPases Rho1 and/or Rho2, stabilize Pck2 and render it competent to exert its biological functions. Remarkably, the target of rapamycin complex 2 (TORC2) does not participate in the catalytic activation of Pck2, but instead contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion. These results unveil a novel mechanism whereby TOR regulates PKC function at a translational level, and they add a new regulatory layer to MAPK signaling cascades.

Engineering Yeast Hexokinase 2 for Improved Tolerance Toward Xylose-Induced Inactivation

Hexokinase 2 (Hxk2p) from Saccharomyces cerevisiae is a bi-functional enzyme being both a catalyst and an important regulator in the glucose repression signal. In the presence of xylose Hxk2p is irreversibly inactivated through an autophosphorylation mechanism, affecting all functions. Consequently, the regulation of genes involved in sugar transport and fermentative metabolism is impaired. The aim of the study was to obtain new Hxk2p-variants, immune to the autophosphorylation, which potentially can restore the repressive capability closer to its nominal level. In this study we constructed the first condensed, rationally designed combinatorial library targeting the active-site in Hxk2p. We combined protein engineering and genetic engineering for efficient screening and identified a variant with Phe159 changed to tyrosine. This variant had 64% higher catalytic activity in the presence of xylose compared to the wild-type and is expected to be a key component for increasing the productivity of recombinant xylose-fermenting strains for bioethanol production from lignocellulosic feedstocks.

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells.

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.

Helix Bundle Loops Determine Whether Histidine Kinases Autophosphorylate in cis or in trans

Bacteria frequently use two-component signal transduction pathways to sense and respond to environmental and intracellular stimuli. Upon receipt of a stimulus, a homodimeric sensor histidine kinase autophosphorylates and then transfers its phosphoryl group to a cognate response regulator. The autophosphorylation of histidine kinases has been reported to occur both in cis and in trans, but the molecular determinants dictating which mechanism is employed are unknown. Based on structural considerations, one model posits that the handedness of a loop at the base of the helical dimerization domain plays a critical role. Here, we tested this model by replacing the loop from Escherichia coli EnvZ, which autophosphorylates in trans, with the loop from three PhoR orthologs that autophosphorylate in cis. These chimeric kinases autophosphorylated in cis, indicating that this small loop is sufficient to determine autophosphorylation mechanism. Further, we report that the mechanism of autophosphorylation is conserved in orthologous sets of histidine kinases despite highly dissimilar loop sequences. These findings suggest that histidine kinases are under selective pressure to maintain their mode of autophosphorylation, but they can do so with a wide range of sequences.

Calcium/Calmodulin Stimulates the Autophosphorylation of Elongation Factor 2 Kinase on Thr-348 and Ser-500 To Regulate Its Activity and Calcium Dependence

Eukaryotic elongation factor 2 kinase (eEF-2K) is an atypical protein kinase regulated by Ca(2+) and calmodulin (CaM). Its only known substrate is eukaryotic elongation factor 2 (eEF-2), whose phosphorylation by eEF-2K impedes global protein synthesis. To date, the mechanism of eEF-2K autophosphorylation has not been fully elucidated. To investigate the mechanism of autophosphorylation, human eEF-2K was coexpressed with λ-phosphatase and purified from bacteria in a three-step protocol using a CaM affinity column. Purified eEF-2K was induced to autophosphorylate by incubation with Ca(2+)/CaM in the presence of MgATP. Analyzing tryptic or chymotryptic peptides by mass spectrometry monitored the autophosphorylation over 0-180 min. The following five major autophosphorylation sites were identified: Thr-348, Thr-353, Ser-445, Ser-474, and Ser-500. In the presence of Ca(2+)/CaM, robust phosphorylation of Thr-348 occurs within seconds of addition of MgATP. Mutagenesis studies suggest that phosphorylation of Thr-348 is required for substrate (eEF-2 or a peptide substrate) phosphorylation, but not self-phosphorylation. Phosphorylation of Ser-500 lags behind the phosphorylation of Thr-348 and is associated with the Ca(2+)-independent activity of eEF-2K. Mutation of Ser-500 to Asp, but not Ala, renders eEF-2K Ca(2+)-independent. Surprisingly, this Ca(2+)-independent activity requires the presence of CaM.

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes.

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.