Preferred Site Of Phosphorylation Research Articles

Ras2 and Ras1 Protein Phosphorylation in Saccharomyces cerevisiae

This work describes the phosphorylation of Saccharomyces cerevisiae Ras proteins and explores the physiological role of the phosphorylation of Ras2 protein. Proteins expressed from activated alleles of RAS were less stable and less phosphorylated than proteins from cells expressing wild-type alleles of RAS. This difference in phosphorylation level did not result from increased signaling through the Ras-cAMP pathway or reflect the primarily GTP-bound nature of activated forms of Ras protein per se. In addition, phosphorylation of Ras protein was not dependent on proper localization of the Ras2 protein to the plasma membrane nor on the interaction of Ras2p with its exchange factor, Cdc25p. The preferred phosphorylation site on Ras2 protein was identified as serine 214. This site, when mutated to alanine, led to promiscuous phosphorylation of Ras2 protein on nearby serine residues. A decrease in phosphorylation may lead to a decrease in signaling through the Ras-cAMP pathway.

Phosphorylation of the High Molecular Weight Neurofilament Protein (NF-H) by Cdk5 and p35

The high molecular weight neurofilament protein (NF-H) is highly phosphorylated in the axon. The phosphorylation sites have been identified as KSP (Lys-Ser-Pro) repeats in the tail domain of NF-H. These KSP sequences are present more than 50 times in the NF-H tail, and most of these sites are normally phosphorylated in vivo. These KSP sites can be further divided into two separate consensus sequences, KSPXK and KSPXY (where Y is not K). The extensive phosphorylation of NF-H has been proposed to play a critical role in the determination of axonal diameter. Recent studies have shown that Cdk5, a kinase related to the cell cycle-dependent kinase Cdc2, is expressed in the brain and associates with the cytoskeleton. In vitro phosphorylation studies have shown that Cdk5 in conjunction with its activator, p35, is able to phosphorylate histone H1, dephosphorylated NF-H, as well as a synthetic peptide with the repetitive KSP motif. We have cloned the cDNAs for rat Cdk5 and p35 by reverse transcription-polymerase chain reaction and cDNA library screening and studied the phosphorylation of NF-H both in vivo and in vitro. By transient transfection assays, we have shown that NF-H can only be extensively phosphorylated in the presence of both Cdk5 and p35. This phosphorylation can be inhibited by a Cdk5-dominant negative mutant, an observation which further supports that Cdk5 is a kinase that is able to phosphorylate NF-H. By immunoprecipitating Cdk5 and p35 from the transfected cells, we have been able to show that the KSPXK repeats are the preferred phosphorylation sites for Cdk5, while the KSPXY repeats are not directly phosphorylated by Cdk5 and p35.

The functional effects of mutations Thr 673 → Asp and Ser 702 → Asp at the Pro-directed kinase phosophorylation sites in the C-terminus of chicken gizzard caldesmon

We expressed the C-terminal 99 amino acids of chicken gizzard caldesmon (658C) and two point mutants in which the preferred phosphorylation sites of MAP kinase and p34 cdc2 kinase, Ser 702 and Thr 673 were altered to aspartic acid. The T673D mutant was indistinguishable from 658C but S702D was not phosphorylated by MAP kinase, was significantly less potent as an inhibitor of actin-tropomyosin activation of myosin MgATPase, and bound less actin-tropomyosin at low concentrations. Thus Ser 702 is involved in the tropomyosin-dependent inhibitory mechanism of caldesmon, and its phosphorylation by MAP kinase or p34 cdc2 kinase could modulate caldesmon function.

Identification of the regulatory site in smooth muscle calponin that is phosphorylated by protein kinase C.

F-actin and tropomyosin inhibited the phosphorylation of calponin by protein kinase C, and the phosphorylation reduced the binding of calponin to F-actin and tropomyosin. Labeled phosphate from [gamma-32P]ATP was retained both on the chymotryptic NH2-terminal 22-kDa fragment, which contains the actin-, tropomyosin-, and calmodulin-binding regions, and on the COOH-terminal 12-kDa fragment. Fractionation of tryptic 32P-labeled peptides by high performance liquid chromatography allowed isolation of three phosphopeptides (designated T1, T2, and T3), each of which was located in three repeating amino acid motifs of calponin. Both the relative initial rates and extent of phosphorylation decreased in the order T2 > T3 > T1. Both serine and threonine residues were phosphorylated in T1 (GASQAGMTAPGTK), and only a threonine residue was phosphorylated in T2 (FASQQGMTAYGTR) and in T3 (GASQQGMTVYGLPR). As the 22-kDa fragment contained only T2, the phosphorylation site in T2 appeared to regulate the binding of calponin to F-actin and tropomyosin. The amino acid sequence of T2 indicates that protein kinase C phosphorylates Thr184. Thus Thr184 is the preferred site of phosphorylation and is functionally the most important of the sites phosphorylated by protein kinase C in smooth muscle calponin.

Inositol (1,4,5)-trisphosphate receptor: characterization of neuron-specific alternative splicing in rat brain and peripheral tissues

One source of diversity in the inositol (1,4,5)-trisphosphate receptors (IP 3Rs) is generated at the level of alternative splicing. Our previous studies of splice isoforms of the receptor in various tissues suggested that some tissues, specifically those containing neurons, selectively express a 40 amino acid insert located between 2 sites for phosphorylation by cyclic AMP-dependent protein kinase (PKA), and that the presence of this insert changes the preferred site of phosphorylation of the receptor. Studies of the mouse receptor have also suggested the existence of intermediately spliced forms containing partial versions of the splice and exhibiting different brain distributions. In this study, we have investigated the alternative splicing of the rat receptor in greater detail using RNase protection and PCR analysis. We find little evidence for the existence of intermediately spliced forms in rat, raising the possibility that the degree of alternative splicing at this site differs in the brains of two very similar species. Our screen of tissue distribution supports the selectively neuronal expression of the long spliced form, and suggests that regulation of this receptor in neurons may be different than in other tissues.

Phosphorylation and modulation of a kainate receptor (GluR6) by cAMP-dependent protein kinase.

Ligand-gated ion channels gated by glutamate constitute the major excitatory neurotransmitter system in the mammalian brain. The functional modulation of GluR6, a kainate-activated glutamate receptor, by adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) was examined with receptors expressed in human embryonic kidney cells. Kainate-evoked currents underwent a rapid desensitization that was blocked by lectins. Kainate currents were potentiated by intracellular perfusion of PKA, and this potentiation was blocked by co-application of an inhibitory peptide. Site-directed mutagenesis was used to identify the site or sites of phosphorylation on GluR6. Although mutagenesis of two serine residues, Ser684 and Ser666, was required for complete abolition of the PKA-induced potentiation, Ser684 may be the preferred site of phosphorylation in native GluR6 receptor complexes. These results indicate that glutamate receptor function can be directly modulated by protein phosphorylation and suggest that a dynamic regulation of excitatory receptors could be associated with some forms of learning and memory in the mammalian brain.

Mechanistic studies on rhodopsin kinase. Light-dependent phosphorylation of C-terminal peptides of rhodopsin.

The phosphorylation of a synthetic peptide, corresponding to the C-terminal 11 amino acids of bovine rhodopsin (VII, residues 338-348), was studied under different conditions. The peptide was only phosphorylated in the presence of photoactivated rhodopsin. Using the same protocol, 12 other peptides, mapping in the rhodopsin C-terminal, were screened for their effectiveness as substrates for rhodopsin kinase. It was found that the peptides became poorer substrates with increasing length, and the best substrates comprised the most C-terminal 9-12 amino acids as opposed to other parts of the C-terminus. It was noted that the absence of the two-terminal residues Pro347 and Ala348 impaired peptide phosphorylation. The effect of the decay of metarhodopsin II on the phosphorylation of rhodopsin and the peptides was determined, and it was found that the rhodopsin and peptide phosphorylations decayed with half times of approximately 33 min and 28 min, respectively. The sites of phosphorylation on the peptides were determined and in all cases the phosphorylation was found to be predominantly on serine residues. Only the 11-residue peptide (VII, residues 338-348) contained significant threonine phosphorylation, which was about 25% that on serine residues. Cumulatively, the results suggest that Ser343 is the preferred site of phosphorylation in vitro. The reason for the poor substrate effectiveness of the larger peptides was examined by competitive experiments in which it was shown that a poorly phosphorylated larger peptide successfully inhibited the phosphorylation of a 'good' peptide substrate. The studies above support a mechanism for rhodopsin kinase that we have termed the 'kinase-activation hypothesis'. This requires that the kinase exists in an inactive form and is activated only after binding to photoactivated rhodopsin.

Analysis of the glycosylation and phosphorylation of the alpha-subunit of the lysosomal enzyme, beta-hexosaminidase A, by site-directed mutagenesis.

The glycosylation and subsequent phosphorylation of mannose residues is a pivotal modification during the biosynthesis of lysosomal enzymes. We have identified the sites of N-linked glycosylation and oligosaccharide phosphorylation on the alpha-subunit of beta-hexosaminidase and have determined the influence of the oligosaccharides on the folding and transport of the enzyme. The potential glycosylation sequences, either singly or in combination, were eliminated through site-directed mutagenesis of the cDNA. By expression of the mutant cDNAs in COS-1 cells, each of the three glycosylation sites on the alpha-subunit was found to be modified by an oligosaccharide. One of the three oligosaccharides was the preferred site of phosphorylation. The absence of any individual oligosaccharide did not diminish the expression of the catalytic activity associated with the alpha-chain, implying proper folding and assembly of subunits. A profound effect was observed, however, when all three oligosaccharides were absent. The unglycosylated alpha-subunit, resulting from genetic alteration of all three glycosylation sites or synthesis of the wild-type protein in the presence of tunicamycin, was catalytically inactive. It was found to be improperly folded into an insoluble aggregate, linked through inappropriate disulfide bonds. The unglycosylated protein was trapped in the lumen of the endoplasmic reticulum and was found in a complex with the Ig heavy chain-binding protein, BiP. The properties of the nonglycosylated, misfolded alpha-subunit were similar to some mutant alpha-subunits in Tay-Sachs disease patients. The results indicate that the oligosaccharides are essential, although not in a site-specific manner, for proper folding and cellular transport of the alpha-subunit.

Substrate recognition determinants of the mitogen-activated 70K S6 kinase from rat liver.

The mitogen-activated 70K S6 kinase has an apparent Km for 40 S ribosomal subunits of 0.25 microM. The apparent Km for a synthetic peptide derived from the carboxyl terminus of S6 and containing all of the in vivo sites of phosphorylation was 2.5-fold higher. A number of shorter peptides revealed that the substrate recognition determinants for the preferred site of phosphorylation, Ser236, reside in a seven-amino acid stretch of S6, residues 231-217. Critical to recognition is a block of 3 consecutive arginines, especially Arg231 and Arg233. In contrast, replacement of Ser235 or the preferred site of phosphorylation, Ser236, with alanine has little effect on the apparent Km. Based on this data the consensus recognition sequence would be Arg-(Arg)-Arg-X-X-Ser-X. A number of kinases known to phosphorylate S6, including cAMP-dependent protein kinase and protein kinase C and the 92K S6 kinase II, were also tested for their ability to phosphorylate a decapeptide containing all the critical recognition determinants. Finally, a synthetic peptide containing a putative 70K S6 kinase autoinhibitory domain did not serve as a substrate for the enzyme but did inhibit its activity, although much less effectively than a synthetic peptide containing all the recognition determinants.

Phosphorylation of caldesmon by p34cdc2 kinase. Identification of phosphorylation sites.

It has recently been shown that caldesmon from non-muscle (Yamashiro, S., Yamakita, Y., Hosoya, H., and Matsumura, F. (1991) Nature 349, 169-172) and smooth muscle cells (Mak, A. S., Watson, M. H., Litwin, C. M. E., and Wang, J. H. (1991) J. Biol. Chem. 266, 6678-6681) can be phosphorylated in vitro by p34cdc2 kinase resulting in the inhibition of caldesmon binding to F-actin and Ca(2+)-calmodulin. In this study, we have identified five phosphorylation sites in smooth muscle caldesmon at Ser582, Ser667, Thr673, Thr696, and Ser702. All the sites bear some resemblance to the S(T)-P-X-X motif recognized by p34cdc2. The preferred site of phosphorylation at Thr673 accounts for about 40% of the total phosphorylation. Four of the sites occur in two pairs of closely spaced sites, Ser667/Thr673 and Thr696/Ser702; phosphorylation of one site in each pair inhibits strongly the phosphorylation of the second site in the same pair, presumably due to the close proximity of the two sites. Similar negative cooperativity in phosphorylation of Ser667 and Thr673 was observed using a 22-residue synthetic peptide containing the two sites. Phosphorylation of Ser667/Thr673 and Thr696/Ser702 account for about 90% of the total level of phosphorylation and these sites are located within the 10-kDa CNBr fragment at the COOH-terminal end of caldesmon known to bind actin and Ca(2+)-calmodulin.

Phosphorylation of chicken cardiac C-protein by calcium/calmodulin-dependent protein kinase II.

Chicken cardiac C-protein was readily phosphorylated by purified calcium/calmodulin-dependent protein kinase II (CaM-kinase II). Maximum incorporation was about 4 mol of 32P/mol of C-protein subunit. Peptide mapping indicated that some of the sites phosphorylated by CaM-kinase II were located on the same phosphopeptides obtained when C-protein was phosphorylated by the cAMP-dependent protein kinase (peptides T1, T2, and T3). There was a fourth peptide (T3a) which was unique to CaM-kinase II phosphorylation. 32P-Amino acid analysis showed that essentially all of the 32P of peptides T1, T2, and T3a was in phosphoserine. cAMP-dependent protein kinase incorporated 32P only into threonine of peptide T3. Threonine was the preferred site of phosphorylation by CaM-kinase II, but there was significant phosphorylation of a serine in peptide T3. Partially purified C-protein preparations contained an associated calcium/calmodulin-dependent protein kinase. Peptide maps obtained from C-protein phosphorylated by the endogenous kinase were similar to those obtained from C-protein phosphorylated by CaM-kinase II. However, the ratio of phosphothreonine to phosphoserine in peptide T3 was lower. This was due to a contaminating phosphatase in the partially purified C-protein which preferentially dephosphorylated the phosphothreonine of peptide T3. It is suggested that the calcium/calmodulin-dependent protein kinase associated with C-protein is similar or identical to CaM-kinase II and that CaM-kinase II may play a role in the phosphorylation of C-protein in the heart.

Phosphorylation of smooth muscle caldesmon by calmodulin-dependent protein kinase II. Identification of the phosphorylation sites.

Smooth muscle caldesmon was phosphorylated by smooth muscle calmodulin-dependent protein kinase II. The extent of phosphorylation obtained was 5.65 mol of phosphate/mol of caldesmon. Phosphorylated protein was subjected to the complete trypsin proteolysis and the produced phosphopeptides were purified by C-8 reverse phase chromatography. Nine phosphopeptides were isolated and by amino acid sequence analysis, eight phosphorylation sites were identified. According to the published amino acid sequence of chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R. G., and Lin, W.-G. (1989) J. Biol. Chem. 264, 13873-13879), these sites were serine 26, serine 59, serine 73, threonine 469, serine 475, serine 587, serine 620, and serine 726. The time course of phosphorylation of these sites was also measured and it was concluded that the first site was serine 73, the second site was serine 26, the third site was serine 726, and the fourth site was serine 587. The preferred phosphorylation sites were located in the amino terminus myosin binding domain whereas slower phosphorylation occurred in the carboxyl terminus actin/calmodulin domain.

Phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases in vitro

We previously demonstrated that protein kinases are useful probes of conformational changes that occur upon photoconversion of phytochrome [Wong, Y.-S., Cheng, H.-C., Walsh, D. A., & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097]. Here we present phosphopeptide analyses of oat phytochrome phosphorylated by three mammalian protein kinases and by a polycation-stimulated, phytochrome-associated protein kinase. Phosphorylation of the Pr form by the cAMP-dependent protein kinase occurs predominantly on Ser17 while Ser598 is the preferred phosphorylation site on Pfr. The cGMP-dependent and Ca2(+)-activated, phospholipid-dependent protein kinases, which phosphorylate only the Pr form of phytochrome, recognize the same region on the phytochrome polypeptide as the cAMP-dependent protein kinase. Polycation-stimulated phytochrome phosphorylation reveals that, in contrast to the mammalian enzymes, the plant kinase recognizes the serine-rich, blocked N-terminus of phytochrome. The potential regulatory role of phytochrome phosphorylation, particularly in the structurally conserved serine/threonine-rich N-terminal region of the phytochrome polypeptide, is suggested by these results.

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.

Significance of the gastrin homology and surrounding sequences in polyomavirus middle T antigen for cell transformation.

Deletion of residues 305 to 327 of polyomavirus middle T antigen, including the (Glu)6-Tyr-315 sequence that is a preferred site of phosphorylation in vitro by pp60c-src, markedly altered viral transformation of rat cells. The efficiency of transformation by the deletion mutant depended on how it was introduced into cells, and the resulting transformants displayed limited growth rates in monolayer and in suspension. Substitution of the polyomavirus residues 305 to 327 with a homologous region (containing [Glu]5-Ala-Tyr) from porcine gastrin did not restore wild-type transforming activity. These mutant middle T antigens interacted with pp60c-src and were phosphorylated in vitro. Thus, although a sequence of consecutive glutamic acid residues followed by a tyrosine is a dominant structural element which strongly influences the physical properties of middle T antigen, its presence did not ensure the biological activity of the protein. Other elements in this region of middle T antigen also contributed substantially to the transforming capacity of polyomavirus.