Phosphorylation of the yeast phospholipid synthesis regulatory protein Opi1p by protein kinase C

Opi1p is a negative regulator of expression of phospholipid-synthesizing enzymes in the yeast Saccharomyces cerevisiae. In this work, we examined the phosphorylation of Opi1p by protein kinase C. Using a purified maltose-binding protein-Opi1p fusion protein as a substrate, protein kinase C activity was time- and dose-dependent, and dependent on the concentrations of Opi1p and ATP. Protein kinase C phosphorylated Opi1p on a serine residue. The Opi1p synthetic peptide GVLKQSCRQK, which contained a protein kinase C sequence motif at Ser(26), was a substrate for protein kinase C. Phosphorylation of a purified S26A mutant maltose-binding protein-Opi1p fusion protein by the kinase was reduced when compared with the wild-type protein. A major phosphopeptide present in purified wild-type Opi1p was absent from the purified S26A mutant protein. In vivo labeling experiments showed that the phosphorylation of Opi1p was physiologically relevant, and that the extent of phosphorylation of the S26A mutant protein was reduced by 50% when compared with the wild-type protein. The physiological consequence of the phosphorylation of Opi1p at Ser(26) was examined by measuring the effect of the S26A mutation on the expression of the phospholipid synthesis gene INO1. The beta-galactosidase activity driven by an INO1-CYC-lacI'Z reporter gene in opi1Delta mutant cells expressing the S26A mutant Opi1p was about 50% lower than that of cells expressing the wild-type Opi1p protein. These data supported the conclusion that phosphorylation of Opi1p at Ser(26) mediated the attenuation of the negative regulatory function of Opi1p on the expression of the INO1 gene.     

Phosphorylation of DARPP-32, a dopamine- and cAMP-regulated phosphoprotein, by casein kinase II

DARPP-32 (dopamine- and cAMP-regulated phosphorprotein, Mr = 32,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is an inhibitor of protein phosphatase-1 and is enriched in dopaminoceptive neurons possessing the D1 dopamine receptor. Purified bovine DARPP-32 was phosphorylated in vitro by casein kinase II to a stoichiometry greater than 2 mol of phosphate/mol of protein whereas two structurally and functionally related proteins, protein phosphatase inhibitor-1 and G-substrate, were poor substrates for this enzyme. Sequencing of chymotryptic and thermolytic phosphopeptides from bovine DARPP-32 phosphorylated by casein kinase II suggested that the main phosphorylated residues were Ser45 and Ser102. In the case of rat DARPP-32, the identification of these phosphorylation sites was confirmed by manual Edman degradation. The phosphorylated residues are located NH2-terminal to acidic amino acid residues, a characteristic of casein kinase II phosphorylation sites. Casein kinase II phosphorylated DARPP-32 with an apparent Km value of 3.4 microM and a kcat value of 0.32 s-1. The kcat value for phosphorylation of Ser102 was 5-6 times greater than that for Ser45. Studies employing synthetic peptides encompassing each phosphorylation site confirmed this difference between the kcat values for phosphorylation of the two sites. In slices of rat caudate-putamen prelabeled with [32P]phosphate, DARPP-32 was phosphorylated on seryl residues under basal conditions. Comparison of thermolytic phosphopeptide maps and determination of the phosphorylated residue by manual Edman degradation identified the main phosphorylation site in intact cells as Ser102. In vitro, DARPP-32 phosphorylated by casein kinase II was dephosphorylated by protein phosphatases-1 and -2A. Phosphorylation by casein kinase II did not affect the potency of DARPP-32 as an inhibitor of protein phosphatase-1, which depended only on phosphorylation of Thr34 by cAMP-dependent protein kinase. However, phosphorylation of DARPP-32 by casein kinase II facilitated phosphorylation of Thr34 by cAMP-dependent protein kinase with a 2.2-fold increase in the Vmax and a 1.4-fold increase in the apparent Km. Phosphorylation of DARPP-32 by casein kinase II in intact cells may therefore modulate its phosphorylation in response to increased levels of cAMP.     

A novel synthetic substrate for casein kinase 2

The nonapeptide DTDSEEEIR, corresponding to amino acid residues 78-86 of calmodulin, was synthesized, and its kinetics of phosphorylation by casein kinase 2 was examined. In the presence of 4 microM polylysine, the phosphorylation rate by casein kinase 2 was 16 times greater than that of synthetic substrate peptide RRREEETEEE reported previously, and almost 1 mol of 32p was incorporated per mol of nonapeptide in 60 min at 37 degrees C. The peptide was not phosphorylated by any other protein kinase. The Thr residue was phosphorylated by casein kinase 2, but Ser was not. The Km value of casein kinase 2 for the nonapeptide was 60 microM, comparable to that of casein, and Vmax for the nonapeptide was 4 times greater than that for casein. Addition of polylysine did not affect the Km value but markedly increased Vmax.     

Phosphorylation of Saccharomyces cerevisiae CTP synthetase at Ser424 by protein kinases A and C regulates phosphatidylcholine synthesis by the CDP-choline pathway

The Saccharomyces cerevisiae URA7-encoded CTP synthetase is phosphorylated and stimulated by protein kinases A and C. Previous studies have revealed that Ser424 is the target site for protein kinase A. Using a purified S424A mutant CTP synthetase enzyme, we examined the effect of Ser424 phosphorylation on protein kinase C phosphorylation. The S424A mutation in CTP synthetase caused a 50% decrease in the phosphorylation of the enzyme by protein kinase C and an 80% decrease in the stimulatory effect on CTP synthetase activity by protein kinase C. The S424A mutation caused increases in the apparent Km values of CTP synthetase and ATP of 20-and 2-fold, respectively, in the protein kinase C reaction. The effect of the S424A mutation on the phosphorylation reaction was dependent on time and protein kinase C concentration. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing Ser424 was a substrate for protein kinase C. Comparison of phosphopeptide maps of the wild type and S424A mutant CTP synthetase enzymes phosphorylated by protein kinases A and C indicated that Ser424 was also a target site for protein kinase C. Phosphorylation of Ser424 accounted for 10% of the total phosphorylation of CTP synthetase by protein kinase C. The incorporation of [methyl-3H]choline into phosphocholine, CDP-choline, and phosphatidylcholine in cells carrying the S424A mutant CTP synthetase enzyme was reduced by 48, 32, and 46%, respectively, when compared with control cells. These data indicated that phosphorylation of Ser424 by protein kinase A or by protein kinase C was required for maximum phosphorylation and stimulation of CTP synthetase and that the phosphorylation of this site played a role in the regulation of phosphatidylcholine synthesis by the CDP-choline pathway.     

Phosphorylation of RGS9-1 by an endogenous protein kinase in rod outer segments

Inactivation of the visual G protein transducin, during recovery from photoexcitation, is regulated by RGS9-1, a GTPase-accelerating protein of the ubiquitous RGS protein family. Incubation of dark-adapted bovine rod outer segments with [gamma-(32)P]ATP led to RGS9-1 phosphorylation by an endogenous kinase in rod outer segment membranes, with an average stoichiometry of 0.2-0.45 mol of phosphates/mol of RGS9-1. Mass spectrometry revealed a single major site of phosphorylation, Ser(475). The kinase responsible catalyzed robust phosphorylation of recombinant RGS9-1 and not of an S475A mutant. A synthetic peptide corresponding to the region surrounding Ser(475) was also phosphorylated, and a similar peptide with the S475A substitution inhibited RGS9-1 phosphorylation. The RGS9-1 kinase is a peripheral membrane protein that co-purifies with rhodopsin in sucrose gradients and can be extracted in buffers of high ionic strength. It is not inhibited or activated significantly by a panel of inhibitors or activators of protein kinase A, protein kinase G, rhodopsin kinase, CaM kinase II, casein kinase II, or cyclin-dependent kinase 5, at concentrations 50 or more times higher than their reported IC(50) or K(i) values. It was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by lowering Ca(2+) to nanomolar levels with EGTA; however, it was not stimulated by the addition of phorbol ester, under conditions that significantly enhanced rhodopsin phosphorylation. A monoclonal antibody specific for the Ser(475)-phosphorylated form of RGS9-1 recognized RGS9-1 in immunoblots of dark-adapted mouse retina. Retinas from light-adapted mice had much lower levels of RGS9-1 phosphorylation. Thus, RGS9-1 is phosphorylated on Ser(475) in vivo, and the phosphorylation level is regulated by light and by [Ca(2+)], suggesting the importance of the modification in light adaptation.     

Phosphorylation at serine 10, a major phosphorylation site of p27(Kip1), increases its protein stability

The association of the p27(Kip1) protein with cyclin and cyclin-dependent kinase complexes inhibits their kinase activities and contributes to the control of cell proliferation. The p27(Kip1) protein has now been shown to be phosphorylated in vivo, and this phosphorylation reduces the electrophoretic mobility of the protein. Substitution of Ser(10) with Ala (S10A) markedly reduced the extent of p27(Kip1) phosphorylation and prevented the shift in electrophoretic mobility. Phosphopeptide mapping and phosphoamino acid analysis revealed that phosphorylation at Ser(10) accounted for approximately 70% of the total phosphorylation of p27(Kip1), and the extent of phosphorylation at this site was approximately 25- and 75-fold greater than that at Ser(178) and Thr(187), respectively. The phosphorylation of p27(Kip1) was markedly reduced when the positions of Ser(10) and Pro(11) were reversed, suggesting that a proline-directed kinase is responsible for the phosphorylation of Ser(10). The extent of Ser(10) phosphorylation was markedly increased in cells in the G(0)-G(1) phase of the cell cycle compared with that apparent for cells in S or M phase. The p27(Kip1) protein phosphorylated at Ser(10) was significantly more stable than the unphosphorylated form. Furthermore, a mutant p27(Kip1) in which Ser(10) was replaced with glutamic acid in order to mimic the effect of Ser(10) phosphorylation exhibited a marked increase in stability both in vivo and in vitro compared with the wild-type or S10A mutant proteins. These results suggest that Ser(10) is the major site of phosphorylation of p27(Kip1) and that phosphorylation at this site, like that at Thr(187), contributes to regulation of p27(Kip1) stability.     

The influence of basic residues on the substrate specificity of protein kinase C

The substrate specificity of protein kinase C has been examined using a series of synthetic peptide analogs of glycogen synthase, ribosomal protein S6, and the epidermal growth factor receptor. The glycogen synthase analog peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala10 was phosphorylated at Ser7 with a Km of 40.3 microM. Peptide phosphorylation was strongly dependent on Arg4. When lysine was substituted for Arg4 the Km was increased approximately 20-fold. Addition of basic residues on either the NH2-terminal or COOH-terminal side of the phosphorylation site of the glycogen synthase peptide improved the kinetics of peptide phosphorylation. The analog Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys-Lys was phosphorylated with a Km of 4.1 microM. Substitution of Ser7 with threonine increased the apparent Km to 151 microM. The truncated peptide Pro1-Leu-Ser-Arg-Thr-Leu-Ser-Val8 was phosphorylated with similar kinetic constants to the parent peptide, however, deletion of Val8 increased the apparent Km to 761 microM. The ribosomal peptide S6-(229-239) was phosphorylated with a Km of approximately 0.5 microM predominantly on Ser236 and is one of the most potent synthetic peptide substrates reported for a protein kinase. The apparent Km for S6 peptide phosphorylation was increased by either deletion of the NH2-terminal 3 residues Ala229-Arg-231 or by substitution of Arg238 on the COOH-terminal side of the phosphorylation site with alanine. This analog peptide, [Ala238]S6-(229-239) was phosphorylated with an approximate 6-fold reduction in Vmax and a switch in the preferred site of phosphorylation from Ser236 to Ser235. These results support the concept that basic residues on both sides of the phosphorylation site can have an important influence on the kinetics of phosphorylation and site specificity of protein kinase C.     

Phosphate groups as substrate determinants for casein kinase I action

Phosphorylation of rabbit muscle glycogen synthase by cyclic AMP-dependent protein kinase has been shown to enhance subsequent phosphorylation by casein kinase I (Flotow, H., and Roach, P. J. (1989) J. Biol. Chem. 264, 9126-9128). In the present study, synthetic peptides based on the sequences of the four phosphorylated regions in muscle glycogen synthase were used to probe the role of substrate phosphorylation in casein kinase I action. With all four peptides, prior phosphorylation significantly stimulated phosphorylation by casein kinase I. A series of peptides was synthesized based on the NH2-terminal glycogen synthase sequence PLSRTLS7VSS10LPGL, in which phosphorylation at Ser7 is required for modification of Ser10 by casein kinase I. The spacing between the P-Ser and the acceptor Ser was varied to have 1, 2, or 3 intervening residues. The peptide with a 2-residue spacing (-S(P)-X-X-S-) was by far the best casein kinase I substrate. When the P-Ser residue at Ser7 was replaced with P-Thr, the resulting peptide was still a casein kinase I substrate. However, substitution of Asp or Glu residues at Ser7 led to peptides that were not phosphorylated by casein kinase I. Phosphorylation of one of the other peptides showed that Thr could also be the phosphate acceptor. From these results, we propose that there are substrates for casein kinase I for which prior phosphorylation is a critical determinant of protein kinase action. In these instances, an important recognition motif for casein kinase I appears to be -S(P)/T(P)-Xn-S/T- with n = 2 much more effective than n = 1 or n = 3. Thus, casein kinase I may be involved in hierarchal substrate phosphorylation schemes in which its activity is controlled by the phosphorylation state of its substrates.     

Determinants on simian virus 40 large T antigen are important for recognition and phosphorylation by casein kinase I.

Casein kinase I has been shown to phosphorylate Ser123 and possibly Thr124, in simian virus 40 (SV40) large T antigen; the same sites are also modified in cultured cells incubated with 32Pi [Friedrich A. Gr?sser, Karl H. Scheidtmann, Polygena T. Tuazon, Jolinda A. Traugh & Gernot Walter (1988) Virology 165, 13-22]. The peptide, A-D-S-Q-H-S-T-P-P, which corresponds to the amino acid sequence 118-125 of SV40 large T antigen, was synthesized together with peptides containing changes in specific amino acid residues on either side of Ser123. These peptides were used as model substrates to determine the amino acids in the SV40 large T antigen important for recognition by casein kinase I. The native peptide identified above, with aspartate at the -4 position, was a poor substrate for casein kinase I in vitro. Peptides with acidic residues added at the -2 and -3 positions, preceding Ser123, were phosphorylated by casein kinase I with apparent Km values around 2 mM and Vmax values up to 500 pmol.min-1.ml-1. When acidic residues were added at both sides of the phosphorylatable serine, the peptide had a first-order rate constant over 20-fold higher than peptides with acidic amino acid residues at the N-terminus only; the apparent Km value was 0.65 mM with a Vmax of 2900 pmol.min-1.ml-1. The effects of modifying Ser120 to phosphoserine were examined by addition of a recognition sequence for the cAMP-dependent protein kinase prior to Ser120. Prior phosphorylation of the peptide at Ser120 lowered the apparent Km to 0.061 mM and increased the Vmax to 360 pmol.min-1.ml-1, a 50-fold decrease in Km for casein kinase I and a 6-fold increase in Vmax as compared to the non-phosphorylated peptide. This indicates that Ser120, which has been shown to be phosphorylated in vivo, provides an appropriate recognition determinant for casein kinase I.     

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells: the role of MAPKs after angiotensin II stimulation

Angiotensin II (AII, 100 nM) stimulation of bovine adrenal chromaffin cells (BACCs) produced angiotensin II receptor subtype 1 (AT1)-mediated increases in extracellular regulated protein kinase 1/2 (ERK1/2) and stress-activated p38MAPK (p38 kinase) phosphorylation over a period of 10 min. ERK1/2 and p38 kinase phosphorylation preceded Ser31 phosphorylation on tyrosine hydroxylase (TOH). The inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) activation, PD98059 (0.1-50 microM) and UO126 (0.1-10 microM), dose-dependently inhibited both ERK2 and Ser31 phosphorylation on TOH in response to AII, suggesting MEK1/2 involvement. The p38 kinase inhibitor SB203580 (20 microM, 30 min) abolished Ser31 and Ser19 phosphorylation on TOH and partially inhibited ERK2 phosphorylation produced by AII. In contrast, 1 microM SB203580 did not affect AII-stimulated TOH phosphorylation, but fully inhibited heat shock protein 27 (HSP27) phosphorylation produced by AII. Also, 1 microM SB203580 fully inhibited Ser19 phosphorylation on TOH and HSP27 phosphorylation in response to anisomycin (30 min, 10 microg/mL). The results suggest that ERKs mediate Ser31 phosphorylation on TOH in response to AII, but p38 kinase is not involved. Previous studies suggesting a role for p38 kinase in the phosphorylation of Ser31 are explained by the non-specific effects of 20 microM SB203580 in BACCs. The p38 kinase pathway is able to phosphorylate Ser19 on TOH in response to anisomycin, but does not do so in response to AII.     

Phosphorylation of serine 833 in cytoplasmic domain of low density lipoprotein receptor by a high molecular weight enzyme resembling casein kinase II

A soluble protein kinase that phosphorylates the last serine residue (Ser-833) in the cytoplasmic domain of the low density lipoprotein (LDL) receptor was purified about 1300-fold from the cytosol of bovine adrenal cortex. The LDL receptor kinase shared several properties with casein kinase II: use of either GTP or ATP; phosphorylation of a typical casein kinase II recognition sequence in the LDL receptor (a serine followed by a cluster of three negatively charged amino acids); and inhibition by heparin. The LDL receptor kinase differed from classic casein kinase II in the following respects: its apparent molecular weight on gel filtration was approximately 500,000 as opposed to the usual molecular weight of 130,000 for casein kinase II; its affinity for the LDL receptor (apparent Km approximately 5 nM) was much greater than its affinity for casein (approximately 10 microM); and its activity was inhibited by polylysine, an agent that stimulates casein kinase II. The physiologic role of this unusual kinase, if any, is unknown.     

DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli.

To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBF1), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBF1 was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylation activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low Km value for GBF1 (220 nM compared to approximately 10 microM for casein) was in the range observed for identified physiological substrates of casein kinase II. Phosphorylation of GBF1 resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.     

The Role of p38 and CK2 Protein Kinases in the Response of RAW 264.7 Macrophages to Lipopolysaccharide

The role of protein kinases p38 and CK2 (casein kinase II) in the response of RAW 264.7 macrophages to the lipopolysaccharide (LPS) from gram-negative bacteria was studied. Using specific p38 and CK2 inhibitors (p38 MAP kinase Inhibitor XI and casein kinase II Inhibitor III, respectively), we investigated the effects of these protein kinases on (i) LPS-induced activation of signaling pathways involving nuclear factor κB (NF-κB), stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and interferon regulatory factor 3 (IRF3); (ii) expression of Toll-like receptor 4 (TLR4) and inducible heat-shock proteins HSP72 and HSP90; and (iii) production of interleukins IL-1α, IL-1β, IL-6, tumor necrosis factor α, and IL-10. Activation of the proapoptotic signaling in the macrophages was evaluated from the ratio between the active and inactive caspase-3 forms and p53 phosphorylation. Six hours after LPS addition (2.5 μg/ml) to RAW 264.7 cells, activation of the TLR4 signaling pathways was observed that was accompanied by a significant increase in phosphorylation of IκB kinase α/β, NF-κB (at both Ser536 and Ser276), p38, JNK, and IRF3. Other effects of macrophage incubation with LPS were an increase in the contents of TLR4, inducible heat-shock proteins (HSPs), and pro- and anti-inflammatory cytokines, as well as slight activation of the pro-apoptotic signaling in the cells. Using inhibitor analysis, we found that during the early response of macrophages to the LPS, both CK2 and p38 modulate activation of MAP kinase and NF-κB signaling pathways and p65 phosphorylation at Ser276/Ser536 and cause accumulation of HSP72, HSP90 and the LPS-recognizing receptor TLR4. Suppression of the p38 MAP kinase and CK2 activities by specific inhibitors (Inhibitor XI and Inhibitor III, respectively) resulted in the impairment of the macrophage effector function manifested as a decrease in the production of the early-response proinflammatory cytokines and disbalance between the pro- and anti-apoptotic signaling pathways leading presumably to apoptosis development. Taken together, our data indicate the inefficiency of therapeutic application of p38 and CK2 inhibitors during the early stages of inflammatory response.     

Casein kinase II accumulation in the nucleolus and its role in nucleolar phosphorylation

A rabbit antiserum against highly purified casein kinase II from mouse tumor cells was used for immunolocalization of the enzyme in fixed, permeabilized mouse cells. Casein kinase II was highly accumulated in nucleoli compared to the extra-nucleolar space of the nucleus or to the cytoplasma. Casein kinase II samples highly purified from the cytoplasma, from the extra-nucleolar fraction of the nucleus or from nucleoli exhibited no differences with respect to structure and function. All samples originally had an alpha 2 beta 2 structure (alpha, 42 kDa; beta, 24 kDa) showing formation of the alpha'-chain (36 kDa) only in the late steps of purification. The isoelectric point of the alpha-chain of all three samples was pH 7.7 and that of the beta-chain was pH 6.4-6.6. Using ATP or GTP, all three casein kinase II samples gave the same results of maximum phosphorylation of purified nucleolar marker phosphoproteins pp105/C23, pp135 and B23, yielding pp135 as one of the most highly phosphorylated proteins with an incorporation of about 75 phosphate groups per molecule pp135. Studies on optimum conditions of phosphorylation of nucleolar phosphoproteins by casein kinase II revealed that each of the protein substrates individually responded to alterations of assay parameters such as pH, magnesium ion and sodium chloride concentrations indicating that predominantly individual structural criteria were responsible for optimum phosphorylation. The determination of the apparent Km of casein kinase II for purified nucleolar phosphoproteins yielded values of 0.15 microM (pp105/C23), 0.1 microM (pp135) and 1.0 microM (B23) identifying them as high-affinity substrates of casein kinase II.     

Ordered multisite protein phosphorylation. Analysis of glycogen synthase kinase 3 action using model peptide substrates

Recognition of substrates by the protein kinase glycogen synthase kinase 3 (GSK-3) usually requires prior phosphorylation of the substrate. Using a peptide based on the glycogen synthase sequence PRPAS(3a)VPPS (3b)PSLS(3c)RHSS(4)PHQS(5)EDEEEP (where the numbers in parentheses denote sites of phosphorylation), we showed previously that phosphorylation of site 5 by casein kinase II was necessary for GSK-3 to phosphorylate the peptide at sites 3a, 3b, 3c, and 4 (Fiol, C. J., Mahrenholz, A. M., Wang, Y., Roeske, R. W., and Roach, P. J. (1987) J. Biol. Chem. 262, 14042-14048). In the present study, variant peptides were synthesized in which sites 3a, 3b, 3c, and 4 were individually replaced by Ala residues (denoted Ala-3c, etc.). All of the variant peptides were substrates for casein kinase II. The peptide Ala-4,Ser(P)-5 was not a substrate for GSK-3 confirming the minimal recognition sequence for the protein kinase as -SXXXS(P)-. The peptides Ala-3c,Ser(P)-5, Ala-3b,Ser(P)-5, and Ala-3a,Ser(P)-5, however, were all good substrates for GSK-3 with apparent Km values in the range 3-6 microns, comparable with that of the parent peptide. GSK-3 could introduce 1, 2, and 3 phosphates, respectively, into these substrates, always COOH-terminal to the substituted Ala residue. Ala-4,Ser(P)-5 and Ala-3c,Ser(P)-4,Ser(P)-5 were competitive inhibitors for phosphorylation of the parent peptide, with Ki values of 2 and 5 microns, respectively. The data suggest (i) that GSK-3 recognizes serines in the motif -SXXXS(P)-, and (ii) that multiple phosphorylation of the peptide substrate has an obligate order, with the sequential formation of new recognition sequences.     

Role of serine 10 phosphorylation in p27 stabilization revealed by analysis of p27 knock-in mice harboring a serine 10 mutation

The inhibition of cyclin-dependent kinase activity by p27 contributes to regulation of cell cycle progression. Serine 10 is the major phosphorylation site of p27, and its phosphorylation has been shown to affect the stability and nuclear export of p27 at the G0-G1 transition in transfected cultured cells. To investigate the physiological relevance of p27 phosphorylation on Ser10, we generated p27 "knock-in" mice that harbor an S10A mutation in this protein. Mice homozygous for the mutation (p27(S10A/S10A) mice) were normal in body size, but the abundance of p27 was decreased in many organs, including brain, thymus, spleen, and testis. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27(S10A/S10A) mice compared with that in wild-type cells, whereas p27 stability in S phase was similar in cells of the two genotypes. The degradation of p27 in cells of the mutant mice at G0 phase was prevented by a proteasome inhibitor. These data indicate that the physiological role of p27 phosphorylation on Ser10 is to stabilize the protein in G0 phase. Unexpectedly, the nuclear export of p27 at the G0-G1 transition occurred normally in p27(S10A/S10A) mouse embryonic fibroblasts, indicating that phosphorylation of Ser10 is dispensable for this process.