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PubMed2006

Phosphorylation of human CTP synthetase 1 by protein kinase A: identification of Thr455 as a major site of phosphorylation.

CTP synthetase is an essential enzyme that generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this study, we examined the phosphorylation of the human CTPS1-encoded CTP synthetase 1 by protein kinase A. CTP synthetase 1 was expressed and purified from a Saccharomyces cerevisiae ura7Delta ura8Delta double mutant that lacks CTP synthetase activity. Using purified CTP synthetase 1 as a substrate, protein kinase A activity was time- and dose-dependent. The phosphorylation, which primarily occurred on a threonine residue, was accompanied by a 50% decrease in CTP synthetase 1 activity. The synthetic peptide LGKRRTLFQT that contains the protein kinase A motif for Thr(455) was a substrate for protein kinase A. A Thr(455) to Ala (T455A) mutation in CTP synthetase 1 was constructed by site-directed mutagenesis and was expressed and purified from the S. cerevisiae ura7Delta ura8Delta mutant. The T455A mutation caused a 78% decrease in protein kinase A phosphorylation and the loss of the phosphothreonine residue and a major phosphopeptide that were present in the purified wild type enzyme phosphorylated by protein kinase A. The CTP synthetase 1 activity of the T455A mutant enzyme was 2-fold higher than the wild type enzyme. In addition, the T455A mutation caused a 44% decrease in the amount of human CTP synthetase 1 that was phosphorylated in S. cerevisiae cells, and this was accompanied by a 2.5-fold increase in the cellular concentration of CTP and a 1.5-fold increase in the choline-dependent synthesis of phosphatidylcholine.

Amino Acid Substitution↗

PubMed1982

Hormone-induced protein phosphorylation. I. Relationship between secretagogue action and endogenous protein phosphorylation in intact cells from the exocrine pancreas and parotid.

We undertook studies to determine whether secretagogue action on the exocrine pancreas and parotid is accompanied by phosphorylation of proteins in intact cells. For this purpose, rat pancreatic, and parotid lobules were preincubated with 32Pi for 45 min at 37 degrees C, washed, and then incubated at 37 degrees C in the presence or absence of secretagogues that effect discharge through different second messengers. Among a variety of polypeptides exhibiting enhanced phosphorylation in pancreatic lobules upon a 30-s incubation in the presence of the secretagogues carbamylcholine, cholecystokinin octapeptide, or secretin, one species with an Mr of 29,000 was especially notable for three reasons: (a) its enhanced level of phosphorylation was dependent on the dose of secretagogue used and was still apparent after incubation for 30 min at 37 degrees C; (b) an analogous phosphorylated polypeptide was observed in isoproterenol-stimulated parotid lobules; and (c) in both tissues its selective dephosphorylation was observed upon termination of stimulation by administration of atropine to carbamylcholine-stimulated pancreatic lobules and propranolol to isoproterenol-stimulated parotid lobules. These results suggest that the phosphorylation of one protein with an Mr of 29,000 is closely correlated both temporally and in a dose-dependent fashion with secretagogue action in both the exocrine pancreas and parotid.

Animals↗

PubMed1989

Phosphorylation of carcinogen metabolizing enzymes: regulation of the phosphorylation status of the major phenobarbital inducible cytochromes P-450 in hepatocytes.

We present data showing that the major phenobarbital inducible cytochromes P-450 (cytochrome P-450IIB1 and cytochrome P-450IIB2) were phosphorylated in intact hepatocytes. This phosphorylation was greatly increased by the cAMP derivatives N6-dibutyryl-cAMP and 8-thiomethyl-cAMP mediated by a cAMP-dependent protein kinase. Most importantly the phosphorylation status of cytochromes P-450 was shown to change in the hepatocytes after treatment with glucagon, which is known to increase the level of cAMP in hepatocytes. The observed impact of the hormone glucagon on the phosphorylation of distinct cytochrome P-450 forms in intact hepatocytes reveals the possibility that the enzyme activity of cytochromes P-450 could be rapidly and differentially regulated by their phosphorylation and therefore dependent on the hormonal status of the organism.

Animals↗

PubMed1999

Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation.

Components of the ras signaling pathway contribute to activation of cellular p53. In MCF-7 cells, p38 kinase activated p53 more effectively than other members of the ras pathway. p53 and p38 kinase exist in the same physical complex, and co-expression of p38 stabilized p53 protein. In vitro, p38 kinase phosphorylated p53 at Ser33 and Ser46, a newly identified site. Mutation of these sites decreased p53-mediated and UV-induced apoptosis, and the reduction correlated with total abrogation of UV-induced phosphorylation on Ser37 and a significant decrease in Ser15 phosphorylation in mutant p53 containing alanine at Ser33 and Ser46. Inhibition of p38 activation after UV irradiation decreased phosphorylation of Ser33, Ser37 and Ser15, and also markedly reduced UV-induced apoptosis in a p53-dependent manner. These results suggest that p38 kinase plays a prominent role in an integrated regulation of N-terminal phosphorylation that regulates p53-mediated apoptosis after UV radiation.

Alanine↗

PubMed1996

Phosphorylated sites of M(r) 25,000 protein, a putative protein phosphatase 2A modulator, and phosphorylation of the synthetic peptide containing these sites by protein kinase C.

The M(r) 25,000 protein isolated from Xenopus laevis oocytes was shown to be an effective phosphate acceptor for Ca(2+)-phospholipid-dependent protein kinase (protein kinase C) [Hashimoto, E. et al. (1995) J. Biochem. 118, 453-460]. In this study, the sites of this protein phosphorylated by protein kinase C were determined and the mechanism of substrate recognition was studied using a synthetic peptide containing the phosphorylation sites. After incorporation of about 2 mol of phosphate per mol of this protein, the radioactive protein was digested with trypsin and the phosphopeptides were purified by a series of column chromatographies. The amino acid sequence of the major radioactive peptide was shown to be Ser-Arg-Val-Ser-Lys-Arg. This and previous results suggest that the two serine residues at the amino-terminal region were phosphorylated by protein kinase C. To confirm this, the phosphorylated protein was directly analyzed for the amino acid sequence. The percent distribution of dithiothreitol adduct of the phenylthiohydantoin derivative of serine (PTH-serine) compared with that of PTH-serine increased at the first and fourth cycles of the sequence analysis. When the synthetic peptide composed of the amino-terminal eleven amino acids was employed as phosphate acceptor, the Km value was unexpectedly high (1.1 mM) compared with that of the native protein (0.5 muM). A stimulatory effect of M(r) 25,000 protein on the activity of protein phosphatase 2A was further enhanced after phosphorylation by protein kinase C. These results suggest that the two serine residues recognized by protein kinase C may have some role in the regulation of this M(r) 25,000 protein.

Amino Acid Sequence↗

PubMed2003

Phosphorylated non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from heterotrophic cells of wheat interacts with 14-3-3 proteins.

Glyceraldehyde-3-phosphate dehydrogenases catalyze key steps in energy and reducing power partitioning in cells of higher plants. Phosphorylated non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) present in heterotrophic cells of wheat (Triticum aestivum) was activated up to 3-fold by MgCl2. The effect was not observed with the non-phosphorylated enzyme found in leaves. The divalent cation also affected the response of the enzyme from endosperm and shoots to adenine nucleotides and inorganic pyrophosphate. Gel filtration chromatography, co-immunoprecipitation followed by immunostaining, and the use of a phosphopeptide containing a canonical binding motif showed that MgCl2 actually disrupted the interaction between GAPN and a 14-3-3 regulatory protein. After interaction with 14-3-3, phosphorylated GAPN exhibits a 3-fold lower Vmax and higher sensitivity to inhibition by ATP and pyrophosphate. Results suggest that GAPN is a target for regulation by phosphorylation, levels of divalent cations, and 14-3-3 proteins. The regulatory mechanism could be critical to maintain levels of energy and reductants in the cytoplasm of heterotrophic plant cells.

14-3-3 Proteins↗

PubMed1983

Synthetic peptides reproducing the site phosphorylated by cAMP-dependent protein kinase in protein phosphatase inhibitor-1. Effect of structural modifications on the phosphorylation efficiency.

The hexapeptide (Arg)2-Pro-Thr-Pro-Ala (T1) and octapeptide (Arg)2-Pro-Thr-Pro-Ala (T5), reproducing the phosphorylatable site of protein phosphatase inhibitor-1, a physiological target of cAMP-dependent protein kinase, and five related peptides were synthesized by the method in solution. The phosphorylation rates of such peptides by cAMP-dependent protein kinase and their kinetic parameters have been determined and compared with those of the hexapeptide (Arg)2-Ala-Ser-Val-Ala, reproducing the phosphorylatable site of rat liver pyruvate kinase. The results obtained show that both the presence of threonine instead of serine and the adjacent C-terminal proline represent highly unfavourable factors seriously impairing the protein kinase reaction by both increasing Km and depressing V. On the other hand the N-terminal proline is compatible with high phosphorylation rates and the row of four rather than two consecutive arginines improves the phosphorylation efficiency by lowering tenfold the Km, without affecting the V. The extension of the hexapeptide T1 on its C-terminal side to give the derivative (Arg)2-Pro-Thr-Pro-Ala-Thr-Val-Ala has no significant effect on the kinetic parameters. Moreover no relationship between the phosphorylation efficiency and the predicted secondary structures around the target residue could be evidenced. Therefore the local structural features of the phosphorylatable site of inhibitor-1 cannot fully account for the fast phosphorylation of this regulatory protein. Other factors must optimize the protein kinase reaction.

Amino Acids↗

PubMed1985

Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Identification of the sites phosphorylated by casein kinase-I.

A casein kinase was highly purified from rabbit skeletal muscle whose substrate specificity and enzymatic properties were virtually identical to those of casein kinase-I from rabbit reticulocytes. Prolonged incubation of glycogen synthase with high concentrations of skeletal muscle casein kinase-I and Mg-ATP resulted in the incorporation of greater than 6 mol phosphate/mol subunit and decreased the activity ratio (+/- glucose-6P) from 0.8 to less than 0.02. The sites phosphorylated by casein kinase-I were all located in the N and C-terminal cyanogen bromide peptides, termed CB-1 and CB-2. At an incorporation of 6 mol phosphate/mol subunit, approximately equal to 2 mol/mol was present in CB-1 and approximately equal to 4 mol/mol in CB-2. Within CB-1, casein kinase-I phosphorylated the serines that were 3, 7 and 10 residues from the N-terminus of glycogen synthase, with minor phosphorylation at threonine-5. Within CB-2, approximately equal to 90% of the phosphate incorporated was located between residues 28 and 53, and at least five of the seven serine residues in this region were phosphorylated. The remaining 10% of phosphate incorporated into CB-2 was located between residues 98 and 123, mainly at a serine residue(s). Two of the major sites labelled by casein kinase-I (serine-3 and serine-10 of CB-1) are not phosphorylated by any other protein kinase. This will enable the role of casein kinase-I as a glycogen synthase kinase in vivo to be evaluated.

Amino Acid Sequence↗

PubMed1997

Effect of phosphorylated rat fetuin on the growth of hepatocytes in primary culture in the presence of human hepatocyte-growth factor. Evidence that phosphorylated fetuin is a natural modulator of hepatocyte-growth factor.

Rat fetuin, a counterpart of human alpha 2-HS glycoprotein and bovine fetuin, that is synthesized and secreted by hepatocytes is mostly phosphorylated, though rat fetuin isolated from bone matrix does not contain phosphorus. A rat 63-kDa phosphorylated N-glycoprotein (pp63) is the phosphorylated form of rat fetuin and pp63 has been shown to inhibit insulin-receptor tyrosine kinase activity. Therefore, we examined the effect of phosphorylated rat fetuin (phosphofetuin) on DNA synthesis in rat hepatocytes in culture in the presence of human hepatocyte-growth factor (HGF), since the human receptor of HGF, c-Met, is known to contain a tyrosine-kinase domain in its intracellular domain. We found that phosphofetuin from conditioned medium of rat-hepatocyte cultures dose-dependently decreased HGF-stimulated DNA synthesis in hepatocytes, whereas addition of non-phosphorylated rat fetuin had no effect. Addition of anti-(rat fetuin) Ig to the culture medium increased HGF-stimulated DNA synthesis by hepatocytes. Immunoprecipitation and cross-linking experiments showed that phosphofetuin bound to human HGF. We found that phosphofetuin interfered with binding of HGF to its specific receptor(s). These observations suggest that phosphofetuin synthesized by hepatocytes may be a natural modulator of HGF as a chalone, and that regulation of expression of phosphofetuin by growth factors and cytokines may be involved in liver regeneration under inflammatory conditions, such as in hepatitis.

Animals↗

PubMed1997

Specific stimulation of c-Fgr kinase by tyrosine-phosphorylated (poly)peptides--possible implication in the sequential mode of protein phosphorylation.

Hematopoietic lineage cell-specific HS1 protein is converted into a substrate for c-Fgr by previous Syk-mediated phosphorylation, at site(s) that bind to the SH2 domain of c-Fgr [Ruzzene, M., Brunati, A. M., Marin, O., Donella-Deana, A. & Pinna, L. A. (1996) Biochemistry 35, 5327-5332]. Here we show that a phosphopeptide derived from one such site, HS1-(320-329)-phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose-dependent manner, the catalytic activity of c-Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c-Fgr itself. The dephosphorylated HS1-(320-329)-peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen-(393-402) sequence, c-Src, and c-Fgr autophosphorylation sites, and the C-terminal c-Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c-Fgr SH2 domain. Stimulation of c-Fgr catalytic activity is also promoted by the full-length HS1 protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased Vmax while the Km values are unchanged. If the normal activator of c-Fgr kinase, Mg2+, is replaced by Mn2+, stimulation by HS1-(320-329)-phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autophosphorylated c-Fgr to be stimulated by HS1-(320-329)-phosphopeptide, support the view that stimulation of c-Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C-terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c-Fgr. These data support the notion that c-Fgr undergoes a unique mechanism of activation promoted by tyrosine-phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein-tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.

Adaptor Proteins, Signal Transducing↗

PubMed1993

Protein kinase FA/GSK-3 phosphorylates tau on Ser235-Pro and Ser404-Pro that are abnormally phosphorylated in Alzheimer's disease brain.

Previously, we identified protein kinase FA/glycogen synthase kinase-3 (GSK-3) as a microtubule-associated protein tau kinase that can incorporate 4 mol of phosphates into 1 mol of tau protein and cause its electrophoretic mobility shift in sodium dodecyl sulfate gels, a unique property characteristic of paired helical filament-associated pathological tau (PHF-tau) in Alzheimer's disease brains. In this report, we identified TPPKS(p)PSAAK and SPVVSGDTS(p)PR as two phosphorylation site sequences phosphorylated by kinase FA/GSK-3 in tau using peptide sequence analysis and sequential manual Edman degradation for radiosequencing. When mapping with human brain tau sequence, we further identified Ser235-Pro and Ser404-Pro as the two major phosphorylation sites according to the numbering of the longest tau isoform. Ser235 and Ser404 have been reported as two of the major abnormal phosphorylation sites in PHF-tau. Taken together, the results provide initial evidence that protein kinase FA/GSK-3 may represent one of the Ser-Pro motif-directed tau kinases involved in the abnormal phosphorylation of pathological PHF-tau in Alzheimer's disease brain.

Alzheimer Disease↗

PubMed1996

Pyrophosphate is a source of phosphoryl groups for Escherichia coli protein phosphorylation.

Pyrophosphate can serve as a source of phosphoryl groups for the phosphorylation of E. coli proteins. The main target of such phosphorylation is a 49-kDa protein which is covalently modified at serine. The same phosphorylation pattern is obtained in the presence of ATP, which suggests that pyrophosphate can substitute for ATP for bacterial protein phosphorylation.

Autoradiography↗

PubMed2003

Phosphorylation of the MAPKKK regulator Ste50p in Saccharomyces cerevisiae: a casein kinase I phosphorylation site is required for proper mating function.

The Ste50 protein of Saccharomyces cerevisiae is a regulator of the Ste11p protein kinase. Ste11p is a member of the MAP3K (or MEKK) family, which is conserved from yeast to mammals. Ste50p is involved in all the signaling pathways that require Ste11p function, yet little is known about the regulation of Ste50p itself. Here, we show that Ste50p is phosphorylated on multiple serine/threonine residues in vivo. Threonine 42 (T42) is phosphorylated both in vivo and in vitro, and the protein kinase responsible has been identified as casein kinase I. Replacement of T42 with alanine (T42A) compromises Ste50p function. This mutation abolishes the ability of overexpressed Ste50p to suppress either the mating defect of a ste20 ste50 deletion mutant or the mating defect of a strain with a Ste11p deleted from its sterile-alpha motif domain. Replacement of T42 with a phosphorylation-mimetic aspartic acid residue (T42D) permits wild-type function in all assays of Ste50p function. These results suggest that phosphorylation of T42 of Ste50p is required for proper signaling in the mating response. However, this phosphorylation does not seem to have a detectable role in modulating the high-osmolarity glycerol synthesis pathway.

Amino Acid Sequence↗

PubMed1999

Characterization of the phosphorylated forms and the phosphorylated residues of hepatitis delta virus delta antigens.

Hepatitis delta virus (HDV) replication requires both the cellular RNA polymerase and one virus-encoded protein, small delta antigen (S-HDAg). S-HDAg has been shown to be a phosphoprotein, but its phosphorylation status is not yet clear. In this study, we employed three methods to address this question. A special two-dimensional gel electrophoresis, namely, nonequilibrium pH gradient electrophoresis, was used to separate the very basic S-HDAg. By carefully adjusting the pH of solubilization solution, the ampholyte composition, and the appropriate electrophoresis time periods, we were able to clearly resolve S-HDAg into two phosphorylated isoforms and one unphosphorylated form. In contrast, the viral large delta antigen (L-HDAg) can only be separated into one phosphorylated and one unphosphorylated form. By metabolic (32)P labeling, both immunoprecipitated S-HDAg and L-HDAg were found to incorporate radioactive phosphate. The extent of S-HDAg phosphorylation was increased upon 12-O-tetradecanoylphorbol-13-acetate treatment, while that of L-HDAg was not affected. Finally, phosphoamino acid analysis identified serine and threonine as the phospho residues in the labeled S-HDAg and only serine in the L-HDAg. Therefore, HDV S- and L-HDAgs differ in their phosphorylation patterns, which may account for their distinct biological functions.

Amino Acid Sequence↗

PubMed2001

Identification of major phosphorylation sites of Epstein-Barr virus nuclear antigen leader protein (EBNA-LP): ability of EBNA-LP to induce latent membrane protein 1 cooperatively with EBNA-2 is regulated by phosphorylation.

Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is a phosphoprotein suggested to play important roles in EBV-induced immortalization of B cells. One of the potential functions of EBNA-LP is a cooperative induction with EBNA-2 of viral and cellular gene expression, including that of the genes for viral latent membrane protein 1 (LMP-1) and cellular cyclin D2. We report here that the phosphorylation of EBNA-LP by cellular kinase(s) is critical to its ability to cooperate with EBNA-2 in up-regulating the expression of LMP-1 in a B-lymphoma cell line. Our conclusion is based on the following observations. (i) Mass-spectrometric analysis of purified EBNA-LP and mutational analyses of EBNA-LP revealed that the serine residue at position 35 in the W2 repeat domain is the major phosphorylation site of EBNA-LP in vivo. (ii) Substitutions of this site in each W2 repeat domain with alanine markedly reduced the ability of the protein to induce LMP-1 expression in combination with EBNA-2 in Akata cells. (iii) Replacement at the major phosphorylation sites with glutamic acids restored the wild-type phenotype. It is well established that this substitution mimics constitutive phosphorylation. These results indicated that the coactivator function of EBNA-LP is regulated by phosphorylation.

Alanine↗

PubMed2003

Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth.

The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.

Active Transport, Cell Nucleus↗

PubMed2006

Phosphorylated HSP27 modulates the association of phosphorylated caldesmon with tropomyosin in colonic smooth muscle.

Thin-filament regulation of smooth muscle contraction involves phosphorylation, association, and dissociation of contractile proteins in response to agonist stimulation. Phosphorylation of caldesmon weakens its association with actin leading to actomyosin interaction and contraction. Present data from colonic smooth muscle cells indicate that acetylcholine induced a significant association of caldesmon with PKCalpha and sustained phosphorylation of caldesmon at ser789. Furthermore, acetylcholine induced significant and sustained increase in the association of phospho-caldesmon with heat-shock protein (HSP)27 with concomitant increase in the dissociation of phospho-caldesmon from tropomyosin. At the thin filament level, HSP27 plays a crucial role in acetylcholine-induced association of contractile proteins. Present data from colonic smooth muscle cells transfected with non-phospho-HSP27 mutant cDNA indicate that the absence of phospho-HSP27 inhibits acetylcholine-induced caldesmon phosphorylation. Our results further indicate that the presence of phospho-HSP27 significantly enhances acetylcholine-induced sustained association of phospho-caldesmon with HSP27 with a concomitant increase in acetylcholine-induced dissociation of phospho-caldesmon from tropomyosin. We thus propose a model whereby upon acetylcholine-induced phosphorylation of caldesmon at ser789, the association of phospho-caldesmon (ser789) with phospho-HSP27 results in an essential conformational change leading to dissociation of phospho-caldesmon from tropomyosin. This leads to the sliding of tropomyosin on actin thus exposing the myosin binding sites on actin for actomyosin interaction.

Acetylcholine↗

PubMed1994

Strong increase in the tyrosine phosphorylation of actin upon inhibition of oxidative phosphorylation: correlation with reversible rearrangements in the actin skeleton of Dictyostelium cells.

When oxidative phosphorylation is inhibited in cells of Dictyostelium discoideum, the phosphorylation of tyrosine residues on actin is strongly increased. This increase is fully reversible. Under the same conditions the amoeboid cells undergo a series of shape changes. Within three minutes the pseudopods are withdrawn and replaced by cell surface blebs. Subsequently, the cells are rounding up to become immobile. In parallel with the changes in cell shape, the distribution of actin filaments is grossly altered within the cells. The cortical network of actin filaments of normal cells is broken down, and the F-actin forms large, irregular clusters deep within the cytoplasm. In these clusters the actin is associated with myosin II and with the heterodimeric F-actin capping protein cap32/34. After restoration of oxidative phosphorylation the actin returns within less than four minutes to its normal cortical position. A causal relationship between tyrosine phosphorylation and changes in the distribution of actin remains to be established. The rearrangements in the actin system that result from the inhibition of oxidative phosphorylation indicate that the organisation of this system and its maintenance in a functional state depend on the continuous supply of energy by ATP.

Actins↗