Phosphorylation of the Epstein-Barr virus nuclear antigen 2.

A major in vivo phosphorylation site of the Epstein-Barr virus nuclear antigen 2 (EBNA-2) was found to be localized at the C-terminus of the protein. In vitro phosphorylation studies using casein kinase 1 (CK-1) and casein kinase 2 (CK-2) revealed that EBNA-2 is a substrate for CK-2, but not for CK-1. The CK-2 specific phosphorylation site was localized in the 140 C-terminal amino acids using a recombinant trpE-C-terminal fusion protein. In a similar experiment, the 58 N-terminal amino acids expressed as a recombinant trpE-fusion protein were not phosphorylated. Phosphorylation of a synthetic peptide corresponding to amino acids 464-476 of EBNA-2 as a substrate led to the incorporation of 0.69 mol phosphate/mol peptide indicating that only one of three potential phosphorylation sites within the peptide was modified. The most likely amino acid residues for phosphorylation by CK-2 are Ser469 and Ser470.     

Polyamines stimulate the activity of glycogen synthase (casein) kinase-1 from bovine kidney and different rat tissues

Previous studies have established that casein kinase-2 (CK-2) is stimulated by polyamines. In this study it is shown that glycogen synthase (casein) kinase-1 (CK-1) can be activated similarly. Using casein as the substrate, bovine kidney CK-1 was stimulated 7-, 2-, and 0.5-fold by spermine, spermidine, and putrescine, respectively. Half-maximal activation of CK-1 by these polyamines was observed at 0.25, 0.70, and 0.50 mM, respectively. CK-1 was optimally activated by spermine at low ionic strength and low Mg2+ concentrations (1-3 mM). Using phosvitin as the substrate, CK-1 was stimulated at low concentrations (0-0.8 mM) and inhibited at higher concentrations of spermine. By contrast CK-2 was inhibited at all concentrations of spermine when phosvitin was used as substrate. Using calcineurin (not a substrate for CK-2) as a substrate, CK-1 from bovine kidney or from three rat tissues (liver, kidney, and testis) was stimulated greater than 2-fold by spermine. It is further shown that heparin inhibits CK-1 and this inhibition can be reversed by spermine. The Vmax of CK-1 for both casein and ATP is increased by spermine while the Km remains unchanged by the polyamine. These studies indicate that CK-1, like CK-2, is a heparin-inhibited and polyamine-activated protein kinase. The results also suggest that CK-1 may be activated by spermine in vivo.     

Non-proline-dependent protein kinases phosphorylate several sites found in tau from Alzheimer disease brain

Of 21 phosphorylation sites identified in PHF-tau 11 are on ser/thr-X motifs and are probably phosphorylated by non-proline-dependent protein kinases (non-PDPKs). The identities of the non-PDPKs and how they interact to hyperphosphorylate PHF-tau are still unclear. In a previous study we have shown that the rate of phosphorylation of human tau 39 by a PDPK (GSK-3) was increased several fold if tau were first prephosphorylated by non-PDPKs (Singh et al., FEBS Lett 358: 267-272, 1995). In this study we have examined how the specificity of a non-PDPK for different sites on human tau 39 is modulated when tau is prephosphorylated by other non-PDPKs (A-kinase, C-kinase, CK-1, CaM kinase II) as well as a PDPK (GSK-3). We found that the rate of phosphorylation of tau 39 by a non-PDPK can be stimulated if tau were first prephosphorylated by other non-PDPKs. Of the four non-PDPKs only CK-1 can phosphorylate sites (thr 231, ser 396, ser 404) known to be present in PHF-tau. Further, these sites were phosphorylated more rapidly and to a greater extent by CK-1 if tau 39 were first prephosphorylated by A-kinase, CaM kinase II or GSK-3. These results suggest that the site specificities of the non-PDPKs that participate in PHF-tau hyperphosphorylation can be modulated at the substrate level by the phosphorylation state of tau.Abbreviations PHF paired helical filaments - A-kinase cyclic AMP-dependent protein kinase - CaM kinase II calcium/calmodulin-dependent protein kinase II - C-kinase calcium/phospholipid-dependent protein kinase - CK-1 casein kinase-1 - CK-2 casein kinase-2 - GSK-3 glycogen synthase kinase-3 - MAP kinase mitogen-activated protein kinase - PDPK proline-dependent protein kinase     

Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.     

Inhibition of glycogen synthase (casein) kinase-1 by heparin

Previous reports have shown that heparin is an inhibitor of casein kinase-2 (CK-2). It is unclear whether heparin is also an inhibitor of glycogen synthase (casein) kinase-1 (CK-1), a type 1 casein kinase. In this study it is shown that CK-1 is potently inhibited by heparin when phosvitin or calcineurin are used as substrates. With casein as a substrate, however, the kinase is insensitive to inhibition by heparin. Using phosvitin as a substrate half-maximal inhibition of CK-1 was observed with 0.14 microgram/ml heparin. Kinetic analyses indicate that at a constant concentration (0.10 mM) of ATP the Km of CK-1 for phosvitin is increased eightfold in the presence of 0.9 microgram/ml heparin; the Vmax is unchanged with or without heparin. At a constant concentration of phosvitin (4 mg/ml) heparin (0.9 microgram/ml) decreased the Vmax for ATP by 57%; the Km is unchanged with or without heparin. The inhibition of CK-1 by heparin can be reversed by KCl (greater than 100 mM). These results indicate that heparin is a potent inhibitor not only of CK-2 but also of CK-1. Hence heparin inhibition can no longer be arbitrarily used as a criterion to discriminate between these kinases.     

Prothymosin alpha is phosphorylated by casein kinase-2.

Prothymosin alpha (ProT alpha) is a 12.5 kDa acidic polypeptide that is considered to have a nuclear function related to cell proliferation. Inspection of its amino acid sequence revealed the presence of sequences that may serve as targets for phosphorylation by casein kinase-2 (CK-2). ProT alpha isolated from calf thymocytes was phosphorylated in vitro by CK-2. The phosphorylation sites are Ser and Thr residues located among the first 14 amino acid residues in the ProT alpha sequence. Another site that is theoretically suitable for phosphorylation by CK-2, at the C-terminus of the polypeptide, is not, in fact, phosphorylated. Thymosin alpha 1 (T alpha 1), a peptide whose sequence corresponds to the first 28 amino acids of ProT alpha, is also phosphorylated by CK-2 at the same phosphorylation sites as ProT alpha. In cultured splenic lymphocytes ProT alpha was phosphorylated at Thr residues located at positions 7, 12 and/or 13. Based on these observations we conclude that CK-2, or another cellular kinase with similar sequence specificity, is responsible for phosphorylation of ProT alpha in vivo.     

Regulation of calcineurin by phosphorylation. Identification of the regulatory site phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C

The site in calcineurin, the Ca2+/calmodulin (CaM)-dependent protein phosphatase, which is phosphorylated by Ca2+/CaM-dependent protein kinase II (CaM-kinase II) has been identified. Analyses of 32P release from tryptic and cyanogen bromide peptides derived from [32P]calcineurin plus direct sequence determination established the site as -Arg-Val-Phe-Ser(PO4)-Val-Leu-Arg-, which conformed to the consensus phosphorylation sequence for CaM-kinase II (Arg-X-X-Ser/Thr-). This phosphorylation site is located at the C-terminal boundary of the putative CaM-binding domain in calcinerin (Kincaid, R. L., Nightingale, M. S., and Martin, B. M. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8983-8987), thereby accounting for the observed inhibition of this phosphorylation when Ca2+/CaM is bound to calcineurin. Since the phosphorylation site sequence also contains elements of the specificity determinants for Ca2+/phospholipid-dependent protein kinase (protein kinase C) (basic residues both N-terminal and C-terminal to Ser/Thr), we tested calcineurin as a substrate for protein kinase C. Protein kinase C catalyzed rapid stoichiometric phosphorylation, and the characteristics of the reaction were the same as with CaM-kinase II: 1) the phosphorylation was blocked by binding of Ca2+/CaM to calcineurin; 2) phosphorylation partially inactivated calcineurin by increasing the Km (from 9.9 +/- 1.1 to 17.5 +/- 1.1 microM 32P-labeled myosin light chain); and 3) [32P]calcineurin exhibited very slow autodephosphorylation but was rapidly dephosphorylated by protein phosphatase IIA. Tryptic and thermolytic 32P-peptide mapping and sequential phosphoamino acid sequence analysis confirmed that protein kinase C and CaM-kinase II phosphorylated the same site.     

Cyclic nucleotide- and Ca2+-independent phosphorylation of tubulin and microtubule-associated protein-2 by glycogen synthase (casein) kinase-1

MAP-2 and tubulin are both shown to be substrates for glycogen synthase (casein) kinase-1 (CK-1). Greater than 40 mol 32P is incorporated into MAP-2 by CK-1 compared to only 14 mol 32P observed when cyclic AMP-dependent protein kinase (A-kinase) is the catalyst. Peptide mapping shows that CK-1 and A-kinase recognize a few common sites; the majority of the sites phosphorylated on MAP-2 by CK-1 are quite distinct. Up to 4 mol 32P can be incorporated into the tubulin dimer by CK-1 compared to only 0.9 mol 32P by A-kinase. The preferred substrate for both kinases is beta-tubulin.     

Regulation of protein phosphatase inhibitor-1 by cyclin-dependent kinase 5

Inhibitor-1, the first identified endogenous inhibitor of protein phosphatase 1 (PP-1), was previously reported to be a substrate for cyclin-dependent kinase 5 (Cdk5) at Ser67. Further investigation has revealed the presence of an additional Cdk5 site identified by mass spectrometry and confirmed by site-directed mutagenesis as Ser6. Basal levels of phospho-Ser6 inhibitor-1, as detected by a phosphorylation state-specific antibody against the site, existed in specific regions of the brain and varied with age. In the striatum, basal in vivo phosphorylation and dephosphorylation of Ser6 were mediated by Cdk5, PP-2A, and PP-1, respectively. Additionally, calcineurin contributed to dephosphorylation under conditions of high Ca2+. In biochemical assays the function of Cdk5-dependent phosphorylation of inhibitor-1 at Ser6 and Ser67 was demonstrated to be an intramolecular impairment of the ability of inhibitor-1 to be dephosphorylated at Thr35; this effect was recapitulated in two systems in vivo. Dephosphorylation of inhibitor-1 at Thr35 is equivalent to inactivation of the protein, as inhibitor-1 only serves as an inhibitor of PP-1 when phosphorylated by cAMP-dependent kinase (PKA) at Thr35. Thus, inhibitor-1 serves as a critical junction between kinase- and phosphatase-signaling pathways, linking PP-1 to not only PKA and calcineurin but also Cdk5.     

Activation of a manganese-dependent membrane protein kinase by serine and tyrosine phosphorylation.

A Mn2(+)-dependent serine/threonine protein kinase from rat liver membranes copurifies with the insulin receptor (IR) on wheat germ agglutinin (WGA)-sepharose. The kinase is present in a nonactivated form in membranes but can be activated 20-fold by phosphorylating the WGA-sepharose fraction with casein kinase-1 (CK-1), casein kinase-2 (CK-2), or casein kinase-3 (CK-3). The activated kinase can use IR beta-subunit, myelin basic protein, and histones as substrates. Activation of the kinase seems to proceed by two or more steps. Sodium vanadate and Mn2+ are required in reaction mixtures for activation to be observed, whereas the tyrosine kinase-specific substrate, poly (glu, tyr), completely inhibits activation. These observations suggest that, in addition to serine/threonine phosphorylation by one of the casein kinases, activation of the Mn2(+)-dependent protein kinase also requires tyrosine phosphorylation. Such phosphorylation may be catalyzed by the IR tyrosine kinase.     

Determination of the site of phosphorylation of nodulin 26 by the calcium-dependent protein kinase from soybean nodules.

Nodulin 26 is a nodule-specific protein that is associated with the symbiosome membrane of soybean root nodules. Nodulin 26 is an endogenous substrate for a novel calcium-dependent protein kinase (CDPK) of soybean root nodules. By phosphopeptide mapping of endoproteinase Lys-C-digested nodulin 26 and automated and manual peptide sequence analyses, we have identified the site on nodulin 26 phosphorylated by CDPK. We have also established that the phosphorylation site of nodulin 26 is identical to the phosphorylation site of CK-15, a synthetic peptide with the carboxyl-terminal sequence of nodulin 26. The phosphorylation of nodulin 26 occurs at position Ser262, and the phosphorylation of CK-15 occurs at the analogous position, Ser,6 in vitro. Thus, the CK-15 sequence apparently contains sufficient structural features of the phosphorylation site of nodulin 26 to be recognized by CDPK. On the basis of peptide mapping analysis of nodulin 26 from nodules that are metabolically labeled with [32P]phosphate, it appears that the site of nodulin 26 that is phosphorylated in vitro is also labeled in vivo. The data indicate that the carboxyl terminus of nodulin 26 is phosphorylated by CDPK and provide initial sequence data for the phosphorylation site of an endogenous substrate for a plant CDPK.     

Phosphotyrosine as a specificity determinant for casein kinase-2, a growth related Ser/Thr-specific protein kinase.

The motif Ser-Ser-Ser-Glu-Glu is readily phosphorylated by casein kinase-2 (CK-2), a growth-related protein kinase whose consensus sequence is Ser(Thr)-Xaa-Xaa-Glu(Asp) [(1990) Biochim. Biophys. Acta 1054, 267-283]. Here we show that phosphotyrosine can replace carboxylic acids as specificity determinant for CK-2 phosphorylation, the phosphotyrosyl peptide Ser-Ser-Ser-TyrP-TyrP actually being a substrate more efficient than Ser-Ser-Ser-Glu-Glu itself both in terms of Km (0.69 vs 2.43 mM) and Vmax. Prior dephosphorylation of phosphotyrosine entirely prevents the subsequent phosphorylation of serine by CK-2. While Ser-Ser-Ser-TyrP-TyrP is a better substrate than Ser-Ser-Ser-SerP-SerP, which in turn is better than Ser-Ser-Ser-Glu-Glu, Ser-Ser-Ser-ThrP-ThrP is a less efficient substrate than Ser-Ser-Ser-Glu-Glu. Thus the order of efficiency of phosphoamino acids as specificity determinants for CK-2 appears to be TyrP greater than SerP much greater than ThrP.     

Identification of peripherin as a Akt substrate in neurons

Activation of Akt-mediated signaling pathways is crucial for survival and regeneration of injured neurons. In this study, we attempted to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt. PC12 cells that overexpressed constitutively active Akt were used. Using two-dimensional PAGE, we identified protein spots that exhibited increased immunostaining of the antibody. Mass spectrometry revealed several major spots as the neuronal intermediate filament protein, peripherin. Using several peripherin fragments, the phosphorylation site was determined as Ser(66) in its head domain in vitro. Furthermore, a co-immunoprecipitation experiment revealed that Akt interacted with the head domain of peripherin in HEK 293T cells. An antibody against phosphorylated peripherin was raised, and induction of phosphorylated peripherin was observed not only in Akt-activated cultured cells but also in nerve-injured hypoglossal motor neurons. These results suggest that peripherin is a novel substrate for Akt in vivo and that its phosphorylation may play a role in motor nerve regeneration.     

Acute calcineurin inhibition with tacrolimus increases phosphorylated UT-A1

UT-A1, the urea transporter present in the apical membrane of the inner medullary collecting duct, is crucial to the kidney's ability to concentrate urine. Phosphorylation of UT-A1 on serines 486 and 499 is important for plasma membrane trafficking. The effect of calcineurin on dephosphorylation of UT-A1 was investigated. Inner medullary collecting ducts from Sprague-Dawley rats were metabolically labeled and treated with tacrolimus to inhibit calcineurin or calyculin to inhibit protein phosphatases 1 and 2A. UT-A1 was immunoprecipitated, electrophoresed, blotted, and total UT-A1 phosphorylation was assessed by autoradiography. Total UT-A1 was determined by Western blotting. A phospho-specific antibody to pser486-UT-A1 was used to determine whether serine 486 can be hyperphosphorylated by inhibiting phosphatases. Inhibition of calcineurin showed an increase in phosphorylation per unit protein at serine 486. In contrast, inhibition of phosphatases 1 and 2A resulted in an increase in UT-A1 phosphorylation but no increase in pser486-UT-A1. In vitro perfusion of inner medullary collecting ducts showed tacrolimus-stimulated urea permeability consistent with stimulated urea transport. The location of phosphorylated UT-A1 in rats treated acutely and chronically with tacrolimus was determined using immunohistochemistry. Inner medullary collecting ducts of the acutely treated rats showed increased apical membrane association of phosphorylated UT-A1 while chronic treatment reduced membrane association of phosphorylated UT-A1. We conclude that UT-A1 may be dephosphorylated by multiple phosphatases and that the PKA-phosphorylated serine 486 is dephosphorylated by calcineurin. This is the first documentation of the role of phosphatases and the specific site of phosphorylation of UT-A1, in response to tacrolimus.     

Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunits.

A number of findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). A recent study has demonstrated that the C tails of the beta and gamma subunits of ENaC are subject to phosphorylation by at least three protein kinases [Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R. & Garty, H. (2002) J. Biol. Chem. 277, 13539-13547]. One of them was identified as ERK which phosphorylates betaT613 and gammaT623 and affects the channel interaction with Nedd4. The current study identifies a second protein kinase as casein kinase 2 (CK2), or CK-2-like kinase. It phosphorylates betaS631, a well-conserved serine on the beta subunit. Such phosphorylation is observed both in vitro using glutathione-S-transferase-ENaC fusion proteins and in vivo in ENaC-expressing Xenopus oocytes. The gamma subunit is weakly phosphorylated by this protein kinase on another residue (gammaT599), and the C tail of alpha is not significantly phosphorylated by this kinase. Thus, CK2 may be involved in the regulation of the epithelial Na+ channel.     

The mengovirus leader protein suppresses alpha/beta interferon production by inhibition of the iron/ferritin-mediated activation of NF-kappa B

In our studies on the biological function of the mengovirus leader protein, we identified a casein kinase II (CK-2) phosphorylation site in the protein. Here we report that the mengovirus leader protein can be phosphorylated by CK-2 in vitro. Expression of a recombinant leader protein in which the consensus CK-2 sequence around threonine 47 was disturbed resulted in a mutant protein that could no longer be phosphorylated. The CK-2 consensus sequence was modified by site-directed mutagenesis and subsequently introduced into a mengovirus cDNA clone to investigate the effect of the phosphorylation of the leader protein on virus replication and on the host cell response. Modifications by which the CK-2 consensus sequence was disturbed resulted in mutant viruses with reduced growth kinetics. We demonstrated that the integrity of the CK-2 phosphorylation site of the mengovirus leader protein was specifically related to the suppression of NF-kappa B activation and subsequent suppression of alpha/beta interferon production in infected cells. We also found that the integrity of the CK-2 phosphorylation site of the leader protein coincided with an increase of ferritin expression in the infected cell. These data indicate that the leader protein suppresses the iron-mediated activation of NF-kappa B and thereby inhibits alpha/beta interferon expression in the infected cell.     

Comparison of the phosphorylation of rabbit skeletal muscle phosphorylase kinase by cAMP-dependent protein kinase and cAMP-independent glycogen synthase (casein) kinase-1

We have previously reported that rabbit skeletal muscle phosphorylase kinase is phosphorylated by glycogen synthase (casein) kinase-1 (CK-1) primarily on the beta subunit (beta = 1 mol of PO4; alpha = 0.2 mol of PO4) when the reaction was carried out in beta-glycerophosphate. The resultant enzyme activation was 16-fold (Singh, T. J., Akatsuka, A., and Huang, K.-P. (1982) J. Biol. Chem. 257, 13379-13384). In the present study we found that in Tris-Cl buffer CK-1 catalyzes the incorporation of greater than 2 mol of PO4/monomer into each of the alpha and beta subunits. Phosphorylase kinase activation resulting from the higher level of phosphorylation remained 16-fold. 32P-Labeled tryptic peptides from the alpha and beta subunits were analyzed by isoelectric focusing. Cyclic AMP-dependent protein kinase (A-kinase) phosphorylates a single major site in each of the alpha and beta subunits at 1.5 mM Mg2+. In addition to these two sites, A-kinase phosphorylates at least three other sites in the alpha subunit at 10 mM Mg2+. CK-1 also catalyzes the phosphorylation of multiple sites in both the alpha and beta subunits. Of the two major sites phosphorylated by CK-1 in the beta subunit, one of these sites is also recognized by A-kinase. At least three sites are phosphorylated by CK-1 in the alpha subunit. One of these sites is recognized by CK-1 only after a prior phosphorylation of phosphorylase kinase by A-kinase at a single site in each of the alpha and beta subunits at 1.5 mM Mg2+. The roles of the different phosphorylation sites in phosphorylase kinase activation are discussed.     

Dephosphorylation of Microtubule-Associated Protein 2, τ Factor, and Tubulin by Calcineurin

Calcineurin dephosphorylated microtubule-associated protein 2 (MAP2) and tau factor phosphorylated by cyclic AMP-dependent and Ca2+, calmodulin-dependent protein kinases from the brain. Tubulin, only phosphorylated by the Ca2+, calmodulin-dependent protein kinase, served as substrate for calcineurin. The concentrations of calmodulin required to give half-maximal activation of calcineurin were 21 and 16 nM with MAP2 and tau factor as substrates, respectively. The Km and Vmax values were in ranges of 1-3 microM and 0.4-1.7 mumol/mg/min, respectively, for MAP2 and tau factor. The Km value for tubulin was in a similar range, but the Vmax value was lower. The peptide map analysis revealed that calcineurin dephosphorylated MAP2 and tau factor universally, but not in a site-specific manner. The autophosphorylated Ca2+, calmodulin-dependent protein kinase was not dephosphorylated by calcineurin. These results suggest that calcineurin plays an important role in the functions of microtubules via dephosphorylation.     

Phosphorylation of Avena phytochrome in vitro as a probe of light-induced conformational changes

A polycation-dependent protein kinase was found to be associated with purified phytochrome preparations from etiolated Avena seedlings. This kinase and three mammalian protein kinases, the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase, and a Ca2+-activated phospholipid-dependent protein kinase, were used to probe light-induced conformational changes in 124-kilodalton Avena phytochrome in vitro. The red absorbing form of phytochrome (Pr) was found to be a substrate for all four protein kinases. Although the far-red absorbing form of phytochrome (Pfr) was as good a substrate as Pr with the cAMP-dependent protein kinase, the Pfr form was poorly phosphorylated by the other three protein kinases. Serine is the major amino acid residue phosphorylated on phytochrome regardless of the form of phytochrome used as substrate. Peptide mapping revealed that the sites of phosphorylation catalyzed by the cAMP-dependent protein kinase differ for Pr and Pfr forms of phytochrome. For the Pr form, the preferred site(s) of phosphorylation was near the amino terminus of the 124-kilodalton subunit. Upon photo-conversion to Pfr, this site can no longer be phosphorylated easily and a new phosphorylation site in the COOH-terminal nonchromophore domain of the molecule becomes accessible to the cAMP-dependent protein kinase. These studies of the phosphorylation of phytochrome provide a new means to study the effect of light absorption by phytochrome on the molecular conformation of the protein. The potential physiological implications of differential phosphorylation of Pr and Pfr await elucidation.     

Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity.

In HeLa cells metabolically labeled in vivo with [32P] orthophosphate in the presence of okadaic acid the concentration of phosphorylated A1 protein was increased significantly as compared to controls. Purified recombinant hnRNP protein A1 served as an excellent substrate in vitro for the catalytic subunit of cAMP-dependent protein kinase (PKA) and for casein kinase II (CKII). Thin layer electrophoresis of A1 acid hydrolysates showed the protein to be phosphorylated exclusively on serine residue by both kinases. V8 phosphopeptide maps revealed that the target site(s) of in vitro phosphorylation are located in the C-terminal region of A1. Phosphoamino acid sequence analysis and site directed mutagenesis identified Ser 199 as the sole phosphoamino acid in the protein phosphorylated by PKA. Phosphorylation introduced by PKA resulted in the suppression of the ability of protein A1 to promote strand annealing in vitro, without any detectable effect on its nucleic acid binding capacity. This finding indicates that phosphorylation of a single serine residue in the C-terminal domain may significantly alter the properties of protein A1.