Differential effects of heparin, fibronectin, and laminin on the phosphorylation of basic fibroblast growth factor by protein kinase C and the catalytic subunit of protein kinase A

Basic fibroblast growth factor (FGF) is synthesized as a phosphoprotein by both bovine capillary endothelial and human hepatoma cells in culture. Because basic FGF is characterized by its high affinity for heparin and its association in vivo with the extracellular matrix, we examined the possibility that the phosphorylation of this growth factor by purified protein kinase C (PK-C) and the catalytic subunit of cAMP- dependent protein kinase-A (PK-A) can be modulated by components of the extracellular matrix. Heparin and other glycosaminoglycans (GAGs) inhibit the ability of PK-C to phosphorylate basic FGF. In contrast, heparin can directly increase the phosphorylation of basic FGF by PK-A. While fibronectin, laminin, and collagen IV have no effect on the ability of PK-C to phosphorylate basic FGF, they all can inhibit the effects of PK-A. Thus, there is a differential effect of extracellular matrix-derived proteins and GAGs on the phosphorylation of basic FGF. The enhanced phosphorylation of basic FGF that is mediated by heparin is associated with a change in the kinetics of the reaction and the identity of the amino acid targeted by this enzyme. The amino acids that are targeted by PK-C and PK-A have been identified by phosphopeptide analyses as Ser64 and Thr112, respectively. In the presence of heparin, basic FGF is no longer phosphorylated by PK-A at the usual PK-A consensus site (Thr112), but instead is phosphorylated at the canonical PK-C site (Ser64). Accordingly, heparin inhibits the phosphorylation of basic FGF by PK-C presumably by masking the PK-C dependent consensus sequence surrounding Ser64. Thus, when basic FGF is no longer phosphorylated by PK-A in the receptor binding domain (Thr112), it loses the increased receptor binding ability that characterizes PK-A phosphorylated basic FGF. The results presented here demonstrate three novel features of basic FGF. First, they identify a functional effect of the binding of heparin to basic FGF. Second, they establish that the binding of heparin to basic FGF can induce structural changes that alter the substrate specificity of protein kinases. Third, and perhaps most important, the results demonstrate the existence of a novel interaction between basic FGF, fibronectin, and laminin. Although the physiological significance of this phosphorylation is not known, these results clearly suggest that the biological activities of basic FGF are regulated by a complex array of biochemical interactions with the proteins, proteoglycans, and glycosaminoglycans present in the extracellular milieu and the cytoplasm.     

Casein kinase II phosphorylates lens connexin 45.6 and is involved in its degradation

Connexin (Cx) 45.6, an avian counterpart of rodent Cx50, is phosphorylated in vivo, but the sites and function of the phosphorylation have not been elucidated. Our peptide mapping experiments showed that the Ser(363) site in the carboxyl (COOH) terminus of Cx45.6 was phosphorylated and that this site is within casein kinase (CK) II consensus sequence, although showing some similarity to CKI sequence. The peptide containing Ser(363) could be phosphorylated in vitro by CKII, but not by CKI. Furthermore, CKII phosphorylated Cx45.6 in embryonic lens membrane and the fusion protein containing the COOH terminus of Cx45.6. Two-dimensional peptide mapping experiments showed that one of the Cx45.6 peptides phosphorylated in vivo migrated to the same spot as one of those phosphorylated by CKII in vitro. Furthermore, CKII activity could be detected in lens lysates. To assess the function of this phosphorylation event, exogenous wild type and mutant Cx45.6 (Ser(363) --> Ala) were expressed in lens primary cultures by retroviral infection. The mutant Cx45.6 was shown to be more stable having a longer half-life compared with wild type Cx45.6. Together, the evidence suggests that CKII is likely a kinase responsible for the Ser(363) phosphorylation, leading to the destablization and degradation of Cx45.6. The connexin degradation induced by phosphorylation has a broad functional significance in the regulation of gap junctions in vivo.     

The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2.

The alpha subunit of eukaryotic protein synthesis initiation factor (eIF-2 alpha) is phosphorylated at a single serine residue (Ser51) by two distinct and well-characterized protein kinase, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51-Arg-Arg-Arg-Ile-Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two-dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP-dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both 'sites' in clupeine Y1 and salmine A1, whereas HCR acted only on the N-terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C-terminal clusters of arginines on the 'target' serine residue, provided that they were present at positions +3 and/or +4 relative to Ser51. However, peptides containing only N-terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF-2 alpha and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.     

The consensus sequences for cdc2 kinase and for casein kinase-2 are mutually incompatible. A study with peptides derived from the beta-subunit of casein kinase-2.

Two series of synthetic peptides that reproduce the amino- and carboxyl-terminal segments of the beta-subunit of casein kinase-2, including the sites phosphorylated by CK2 and cdc2 kinase, respectively, have been used as model substrates for these enzymes. The N-terminal peptide beta(1-9), MSSSEEVSW, is readily phosphorylated by CK2 but not all by cdc2. The opposite is true of the C-terminal peptide beta(206-215), NFKSPVKTIR, whose Ser-4 is a good target for cdc2 while being unaffected by CK2. The individual substitutions of Pro-5 and Lys-7 in the latter peptide with Gly and Ala (or Glu), respectively, prevent its phosphorylation by cdc2, whereas the substitution of Lys-3 with Ala is well tolerated and the substitution of the target Ser with Thr actually improves phosphorylation. Thus the consensus sequence for cdc2 is shown to be X-S-P-X-K. Such a requirement for a basic residue at position +3 is opposite to that of CK2 whose consensus sequence (S-X-X-E/D/Yp/Sp) includes an acidic residue at the same position. Moreover the motif Ser-Pro is detrimental for CK2, preventing the phosphorylation of otherwise suitable peptides. These observations would rule out the possibility that the site specificity of CK2 might overlap with that of cdc2 and possibly of other Pro-directed protein kinases.     

Sequence of sites on ATP-citrate lyase and phosphatase inhibitor 2 phosphorylated by multifunctional protein kinase (a glycogen synthase kinase 3 like kinase)

Multifunctional protein kinase (MFPK) phosphorylates ATP-citrate lyase on peptide B on two sites, BT and BS, on threonine and serine, respectively, inhibitor 2 on a threonyl residue, and glycogen synthase at sites 2 and 3. The phosphorylation sites BT and BS of ATP-citrate lyase are dependent on prior phosphorylation at site A whereas site A phosphorylation is decreased by prior phosphorylation at sites BT and BS. To study the MFPK recognition sites and the site-site interactions, the amino acid sequences of ATP-citrate lyase peptide B and inhibitor 2 were determined and compared to each other and to glycogen synthase sites 3-5. The sequence of the tryptic peptide containing the two phosphorylation sites of peptide B is -Phe-Leu-Leu-Asn-Ala-Ser-Gly-Ser-Thr-Ser-Thr(P)-Pro-Ala-Pro-Ser(P)-Arg-, and the sequence of the MFPK phosphorylation site of inhibitor 2 is -Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-. This inhibitor 2 site is identical with the site phosphorylated by glycogen synthase kinase 3/FA. These results suggest that at least some of the sites phosphorylated by MFPK (BT of ATP-citrate lyase, Thr 72 of inhibitor 2, and sites 3b and 4 of glycogen synthase) contain a Ser/Thr flanked by a carboxyl-terminal proline. However, as MFPK did not phosphorylate a series of peptides containing the -X-Thr/Ser-Pro-X- sequence, this minimum consensus sequence is not sufficient for phosphorylation by MFPK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.     

Chemical genetics approach to identify peptide ligands that selectively stimulate DAPK-1 kinase activity

Dissection of signal transduction pathways has been advanced by classic genetic approaches including targeted gene deletion and siRNA-based inhibition of gene product synthesis. Chemical genetics is a biochemical approach to develop small peptide-mimetic ligands to alter, post-translationally, how an enzyme functions. DAPK-1 was used as a model enzyme to develop selective peptide ligands that modulate its specific activity. The tumor modifier p21 has the most highly conserved elements of a DAPK consensus substrate, including a basic core followed by a hydrophobic core. Therefore, the p21 protein was synthesized in overlapping fragments to acquire a panel of peptide ligands for testing in DAPK binding and phosphorylation assays. Three distinct p21 derived peptide fragments were found to bind to DAPK; however, these had no stimulatory effect on its activity toward in vivo substrates, p21 and MLC. The p21 peptide ligands did, however, strikingly stimulate DAPK activity toward p53, a substrate that shows conservation in the hydrophobic part of its DAPK-1 consensus site. DAPK-1 stimulatory peptides attenuate tryptic cleavage of DAPK-1, suggesting that ligand binding can alter DAPK-1 conformation and lock the enzyme onto its substrate. We, therefore, generated an artificial p53, containing arginine residues N-terminal to the phospho-acceptor site, creating a better DAPK-1 peptide consensus and demonstrated that the Km for p531-66[ET-->RR] and ATP is elevated. The full-length p53E17T18-->R17R18 also functioned as a better Ser20 kinase substrate in vivo. These data suggest that DAPK-1 binding ligands can be generated to elevate its specific activity toward weak substrates and provide an approach to develop genetic assays to alter DAPK-1-specific activity in vivo.     

The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2.

Cyclin D-Cdk4/6 and cyclin A/E-Cdk2 are suggested to be involved in phosphorylation of the retinoblastoma protein (pRB) during the G1/S transition of the cell cycle. However, it is unclear why several Cdks are needed and how they are different from one another. We found that the consensus amino acid sequence for phosphorylation by cyclin D1-Cdk4 is different from S/T-P-X-K/R, which is the consensus sequence for phosphorylation by cyclin A/E-Cdk2 using various synthetic peptides as substrates. Cyclin D1-Cdk4 efficiently phosphorylated the G1 peptide, RPPTLS780PIPHIPR that contained a part of the sequence of pRB, while cyclins E-Cdk2 and A-Cdk2 did not. To determine the phosphorylation state of pRB in vitro and in vivo, we raised the specific antibody against phospho-Ser780 in pRB. We confirmed that cyclin D1-Cdk4, but not cyclin E-Cdk2, phosphorylated Ser780 in recombinant pRB. The Ser780 in pRB was phosphorylated in the G1 phase in a cell cycle-dependent manner. Furthermore, we found that pRB phosphorylated at Ser780 cannot bind to E2F-1 in vivo. Our data show that cyclin D1-Cdk4 and cyclin A/E Cdk2 phosphorylate different sites of pRB in vivo.     

Synthesis of complex phosphopeptides as mimics of p53 functional domains.

A complete set of mono-, di- and triphosphorylated peptides comprising amino acids 10-27, the Mdm2 and p300 binding site(s) of p53, with and without a fluorescein label at the N-terminus, was synthesized by step-by-step solid phase synthesis. Fluorescence polarization analysis revealed that phosphorylation at Thr18 decreased binding to recombinant Mdm2 protein compared with the unphosphorylated and the two other single phosphorylated analogues. Unlabelled multiply phosphorylated peptides corresponding to this amino-terminal transactivation domain proved to be powerful tools in analysing the phosphate specificity of existing anti-p53 monoclonal and polyclonal antibodies using direct ELISA. The tetramerization domain of human p53 protein was modelled with a 53 residue-long unlabelled unphosphorylated and Ser315-phosphorylated peptide pair. CD analysis showed similar alpha-helical structures for both peptides and no major difference in the secondary structure could be observed upon phosphorylation. Size-exclusion HPLC indicated that these synthetic oligomerization domain mimics underwent a pH-dependent tetramerization process, but the presence of a phosphate group at Ser315 did not modify the oligomeric state of the 308-360 p53 fragments. Nevertheless, the fluorescein-labelled Ser315 phosphorylated peptide bound to the downstream signalling ligand DNA topoisomerase I protein with slightly higher affinity than did the unphosphorylated analogue.     

The regulatory beta subunit of protein kinase CK2 contributes to the recognition of the substrate consensus sequence. A study with an eIF2 beta-derived peptide

CK2 is a ubiquitous and pleiotropic Ser/Thr-specific protein kinase that phosphorylates more than 300 protein substrates at sites specified by an acidic consensus sequence in which positions n + 3 and n + 1 are particularly important. Recognition of substrates by CK2 is known to rely on basic residues located in the catalytic site of the alpha subunit which make electrostatic contacts with the negative charges in the substrate consensus sequence, thereby assuring optimal binding; the regulatory beta subunit is believed to play a protective and stabilizing role. We describe a biochemical and structural analysis of CK2-mediated phosphorylation of a 22-mer synthetic peptide corresponding to the N-terminal tail of the eukaryotic translation initiation factor eIF2beta. Results demonstrate that this peptide still displays phosphorylation features similar to full-length eIF2beta and the CK2 beta subunit also contributes to recognition of the protein substrate by establishing both polar and hydrophobic interactions with specificity determinants located downstream from the phosphoacceptor site. In particular, the N-terminal domain of the beta subunit appears to be of crucial importance for optimizing high-affinity phosphorylation of the eIF2beta peptide. This domain includes an acidic cluster whose electrostatic contacts with basic residues of the substrate attenuate intrasteric pseudosubstrate inhibition while strengthening substrate-kinase binding.     

Identification of the major phosphorylation site of the hepatitis C virus H strain NS5A protein as serine 2321.

The hepatitis C virus (HCV) NS5A protein is phosphorylated by a cellular, serine/threonine kinase. To identify the major site(s) of NS5A phosphorylation, radiolabeled HCV-H NS5A phosphopeptides were purified and subjected to phosphoamino acid analysis and Edman degradation. These data identified the major intracellular phosphorylation site in the HCV-H NS5A protein as Ser(2321), a result verified by two additional, independent methods: (i) substitution of Ala for Ser(2321) and the concomitant disappearance of the major in vivo phosphorylated peptides and corresponding in vitro phosphorylated peptides; and (ii) comigration of the digestion products of a synthetic peptide phosphorylated on Ser(2321) with the major in vivo phosphorylated NS5A peptides. Site-directed mutagenesis of Ser(2321) suggested that phosphorylation of NS5A is dispensable for previously described interactions with NS4A and PKR, a cellular, antiviral kinase that does not appear to catalyze NS5A phosphorylation. The proline-rich nature of the amino acid sequence flanking Ser(2321) (PLPPPRS(2321) PPVPPPR) suggests that a proline-directed kinase is responsible for the majority of HCV NS5A phosphorylation, consistent with previous kinase inhibitor studies.     

Specificity determinants of substrate recognition by the protein kinase DYRK1A

DYRK1A is a dual-specificity protein kinase that is thought to be involved in brain development. We identified a single phosphorylated amino acid residue in the DYRK substrate histone H3 (threonine 45) by mass spectrometry, phosphoamino acid analysis, and protein sequencing. Exchange of threonine 45 for alanine abolished phosphorylation of histone H3 by DYRK1A and by the related kinases DYRK1B, DYRK2, and DYRK3 but not by CLK3. In order to define the consensus sequence for the substrate specificity of DYRK1A, a library of 300 peptides was designed in variation of the H3 phosphorylation site. Evaluation of the phosphate incorporation into these peptides identified DYRK1A as a proline-directed kinase with a phosphorylation consensus sequence (RPX(S/T)P) similar to that of ERK2 (PX(S/T)P). A peptide designed after the optimal substrate sequence (DYRKtide) was efficiently phosphorylated by DYRK1A (K(m) = 35 microM) but not by ERK2. Both ERK2 and DYRK1A phosphorylated myelin basic protein, whereas only ERK2, but not DYRK1A, phosphorylated the mitogen-activated protein kinase substrate ELK-1. This marked difference in substrate specificity between DYRK1A and ERK2 can be explained by the requirement for an arginine at the P -3 site of DYRK substrates and its presumed interaction with aspartate 247 conserved in all DYRKs.     

Identification of phosphorylation sites on murine nuclear lamin C by RP-HPLC and microsequencing

Isolated interphase lamin C, obtained from Ehrlich ascites tumor cells, was digested by Lys-C endoproteinase, the resulting peptides separated by reversed-phase HPLC and subjected to microsequencing in order to identify phosphorylation sites in interphase and following phosphorylation in vitro by cdc2-kinase, protein kinase C (PKC) and protein kinase A (PKA), respectively. Nuclear lamin C showed partial phosphorylation of Ser392 and Ser409, and possibly Ser407 in interphase. Phosphorylation was increased in response to cdc2-kinase at Ser390 and Ser392 and to PKC at Ser572. The N-terminal peptide (aa 1-32) containing consensus sequences for the 3 kinases was phosphorylated by cdc2-kinase, PKC and PKA. The sequence data suggests that multiple molecular switches via lamina modification control the dynamic behaviour of the nucleoskeleton during the cell cycle.     

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.     

The phosphorylation sites of troponin T from white skeletal muscle and the effects of interaction with troponin C on their phosphorylation by phosphorylase kinase.

1. The phosphorylation of troponin T from rabbit white sketetal muscle is catalysed by phosphorylase kinase, but not at a significant rate by bovine 3':5'-cyclic AMP-dependent protein kinase. 2. The amino acid sequences adjacent to the three major phosphorylation sites of troponin T were determined. 3. The serine in the N-terminal peptide (Asx,SerP, Glx)Glu-Val-Glu, is that phosphorylated (SerP, phosphoserine) when the troponin complex is isolated. 4. The other two sites of phosphorylation are located in the sequence Ala-Leu-(Ser, SerP)-Met-Gly-Ala-Asn-Tyr(Ser,SerP)Tyr. 5. When troponin T is phosphorylated in the presence of troponin C, the extent of phosphorylation at each site is considerably decreased. 6. CNBr fragments of troponin T are also phosphorylated by phosphorylase kinase, but the rate of phosphorylation at each site in the CNBr fragments is considerably slower than in the native protein. 7. From these studies it is suggested that troponin C interacts with troponin T in the region containing the two closely situated phosphorylation sites.     

Role of phosphorylated aminoacyl residues in generating atypical consensus sequences which are recognized by casein kinase-2 but not by casein kinase-1.

Casein kinase-2 (CK-2) is a ubiquitous Ser/Thr specific protein kinase that recognizes phosphorylatable residues located upstream of acidic determinants, its consensus sequence being Ser(Thr)-Xaa-Xaa-Acidic. Here we show that the phosphotetrapeptide AcSer(P)-Ser(P)-Ser-Ser(P), which is devoid of the canonical consensus sequence, is nevertheless phosphorylated by CK-2 with rates comparable to that of typical peptide substrates Ser-Glu-Glu-Glu-Glu-Glu and Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu routinely employed for assaying CK-2 activity. The phosphopeptide AcSer(P)-Ser-Ser(P) [but not Ac-Ser-Ser(P)-Ser(P) or AcSer(P)-Ser(P)-Ser] is also phosphorylated albeit less efficiently than AcSer(P)-Ser(P)-Ser-Ser(P). Further N-terminal elongation with additional phosphoseryl residues to give the peptides AcSer(P)-Ser(P)-Ser(P)-Ser-Ser(P) and AcSer(P)-Ser(P)-Ser(P)-Ser(P)-Ser-Ser(P) does not improve but rather slightly decreases the phosphorylation efficiency by CK-2. These two peptides are conversely excellent substrates for CK-1, which does not appreciably phosphorylate either AcSer(P)-Ser-Ser(P) or AcSer-(P)-Ser(P)-Ser-Ser(P). Either individual or multiple replacement of the phosphorylated residues with glutamic acid in the peptide AcSer(P)-Ser(P)-Ser-Ser(P) drastically reduces the phosphorylation efficiency by CK-2, the phosphoseryl residue at position -2 playing an especially crucial role which cannot be surrogated by glutamyl residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of synthetic peptides by a CDPK and plant SNF1-related protein kinase. Influence of proline and basic amino acid residues at selected positions.

Spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) can be inactivated by phosphorylation of Ser-158 by calmodulin-like domain protein kinases (CDPKs) or SNF1-related protein kinases (SnRK1) in vitro. While the phosphorylation site sequence is relatively conserved, most of the deduced sequences of SPS from dicot species surrounding the Ser-158 regulatory phosphorylation site contain a Pro residue at P-4 (where P is the phosphorylated Ser); spinach is the exception and contains an Arg at P-4. We show that a Pro at P-4 selectively inhibits phosphorylation of the peptide by a CDPK relative to a SnRK1. The presence of a Pro at P-4, by allowing a tight turn in the peptide substrate, may interfere with proper binding of residues at P-5 and beyond. Both kinases had greater activity with peptides having basic residues at P-6 and P+5 (in addition to the known requirement for an Arg at P-3/P-4), and when the residue at P-6 was a His, the pH optimum for phosphorylation of the peptide was acid shifted. The results are used to predict proteins that may be selectively phosphorylated by SnRK1s (as opposed to CDPKs), such as SPS in dicot species, or may be phosphorylated in a pH-dependent manner.     

Phorbol Myristate Acetate and 8-Bromo-Cyclic AMP-Induced Phosphorylation of Glial Fibrillary Acidic Protein and Vimentin in Astrocytes: Comparison of Phosphorylation Sites

Both the protein kinase C (PK-C) activator, phorbol 12-myristate 13-acetate (PMA), and the cyclic AMP-dependent protein kinase (PK-A) activator, 8-bromo-cyclic AMP (8-BR), have been shown to increase 32P incorporation into glial fibrillary acidic protein (GFAP) and vimentin in cultured astrocytes. Also, treatment of astrocytes with PMA or 8-BR results in the morphological transformation of flat, polygonal-shaped cells into stellate, process-bearing cells, suggesting the possibility that signals mediated by these two kinase systems converge at the level of protein phosphorylation to elicit similar changes in cell morphology. Therefore, studies were conducted to determine whether treatment with PMA and 8-BR results in the phosphorylation of the same tryptic peptide fragments on GFAP and vimentin in astrocytes. Treatment with PMA increased 32P incorporation into all the peptide fragments that were phosphorylated by 8-BR on both vimentin and GFAP; however, PMA also stimulated phosphorylation of additional fragments of both proteins. The phosphorylation of vimentin and GFAP resulting from PMA or 8-BR treatment was restricted to serine residues in the N-terminal domain of these proteins. Studies were also conducted to compare the two-dimensional tryptic phosphopeptide maps of GFAP and vimentin from intact cells treated with PMA and 8-BR with those produced when the proteins were phosphorylated with purified PK-C or PK-A. PK-C phosphorylated the same fragments of GFAP and vimentin that were phosphorylated by PMA treatment. Additionally, PK-C phosphorylated some tryptic peptide fragments of these proteins that were not observed with PMA treatment in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the Phosphate Group with Different Negative Charges on the Conformation of Phosphorylated Ser/Thr-Pro Motif

Summary Ser/Thr-Pro motif is a widespread phosphorylated site in proteins, and its reversible phosphorylation is an important regulatory progress in many cell cycles and signal transduction. Recent research reveals that phosphorylation affects the local conformation of the peptide and its binding with the substrate through peptidyl--prolyl cis/trans isomerization. In order to further explore the effect of the phosphate group with different charges, four model peptides containing non- and phosphorylated Ser/Thr-Pro motif were synthesized using the classical solid-phase method. 1H-NMR, TOCSY, and ROESY were employed to characterize the conformation of the model peptides in solution with different pH value and analyze the peptidyl--prolyl isomerization at a molecular level. The results demonstrate that phosphorylation increases the cis conformation in the peptide and the maximum cis/trans ratio is obtained when the phosphate group has two negative charges. Furthermore, the experiments prove that the phosphorylation introduces a hydrogen bond between the phosphate and the NH of Ser/Thr residue, and the charges of the phosphate affect certain conformations of the phosphorylated Ser/Thr-Pro motif.