Cdc42-independent activation and translocation of the cytostatic p21-activated protein kinase gamma-PAK by sphingosine.

Autophosphorylation of p21-activated protein kinase gamma-PAK is stimulated at 10 microM sphingosine in vitro and is maximal at 100 microM. Sites autophosphorylated on gamma-PAK in response to sphingosine are identical to those obtained with Cdc42(GTP). Autophosphorylation is paralleled by stimulation of gamma-PAK activity as measured with peptide and protein substrates. In 3T3-L1 cells, sphingosine stimulates the autophosphorylation and activity of gamma-PAK associated with the membrane-containing particulate fraction by 2.8-fold, but does not stimulate the activity of the soluble enzyme. Thus, gamma-PAK is activatable via a Cdc42-independent mechanism, suggesting sphingosine has a role in gamma-PAK activation under conditions of cell stress.

Functional interaction between c-Abl and the p21-activated protein kinase gamma-PAK.

A member of the p21-activated protein kinase (PAK) family, gamma-PAK has cytostatic properties and is activated by cellular stresses such as hyperosmolarity or DNA damage. We report herein that gamma-PAK is associated in vivo with the nonreceptor protein tyrosine kinase c-Abl. gamma-PAK phosphorylates c-Abl on sites located in the kinase domain, in a region that is implicated in protein-protein interactions and in subcellular localization. Activation of gamma-PAK in human embryonic kidney 293T cells by cotransfection with constitutively active Cdc42 induces activation of c-Abl, resulting in increased phosphotyrosine levels. Cotransfection of c-Abl and gamma-PAK elicits phosphorylation of gamma-PAK on tyrosine and down-regulation of gamma-PAK activity, promoting accumulation of inactive gamma-PAK. gamma-PAK is also phosphorylated in vitro by c-Abl. gamma-PAK activity is regulated by ubiquitination and proteolysis in vivo, as shown by immunoblotting with an anti-ubiquitin antibody in the presence of proteasome inhibitors. In summary, we describe a functional interaction between gamma-PAK and c-Abl in which gamma-PAK stimulates c-Abl tyrosine kinase activity and c-Abl phosphorylates and down-regulates gamma-PAK, suggesting the existence of a negative feedback loop between c-Abl and gamma-PAK.

Substrates enhance autophosphorylation and activation of p21-activated protein kinase gamma-PAK in the absence of activation loop phosphorylation.

The p21-activated protein kinase gamma-PAK from rabbit, expressed in insect cells, is activated following binding of Cdc42(GTPgammaS). The rate of autophosphorylation is increased fivefold and the protein kinase activity 13-fold, as measured with the synthetic heptapeptide (AKRESAA). The mutant K278R, where the invariant lysine in the catalytic site is replaced by arginine, shows neither autophosphorylation nor activity. Replacement of the conserved threonine in the catalytic domain with alanine (T402A) reduces autophosphorylation and protein kinase activity to 1% that of the wild-type gamma-PAK, indicating autophosphorylation of Thr402 in the activation loop is essential for protein kinase activity. In contrast, certain protein substrates such as histone 2B, histone 4 and myelin basic protein, stimulate both autophosphorylation and protein kinase activity to levels similar to those observed with Cdc42(GTPgammaS). This substrate-level activation does not require autophosphorylation of Thr402 in the activation loop. As shown with T402A, the protein kinase activity with histone 4 is similar to that observed with recombinant wild-type gamma-PAK. Basic proteins or peptides which are not substrates of gamma-PAK, such as histone 1 and polylysine, do not stimulate autophosphorylation or activity. Other substrates such as the Rous sarcoma virus protein NC are phosphorylated by gamma-PAK following activation by Cdc42(GTPgammaS), but are not phosphorylated by T402A. The data suggest that some substrates can override the requirement for Cdc42(GTPgammaS), by activating gamma-PAK directly.

Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation.

p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous gamma-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma-PAK, while native gamma-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of gamma-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, gamma-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma-PAK, six of which are in common with those previously reported in alpha-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma-PAK activation.

Autophosphorylation and protein kinase activity of p21-activated protein kinase gamma-PAK are differentially affected by magnesium and manganese.

To examine the requirements for activation of the p21-activated protein kinase gamma-PAK (Pak2, PAK I) from rabbit reticulocytes by Cdc42(GTPgammaS), autophosphorylation with ATP(Mg) or ATP(Mn) and its effects on protein kinase activity were examined. Autophosphorylation with ATP(Mg) alone was minimal with negligible protein kinase activity; the rate of autophosphorylation was increased 3-4-fold upon binding of Cdc42(GTPgammaS), resulting in a 3-fold stimulation of protein kinase activity with peptide and protein substrates. The rate of autophosphorylation with ATP(Mn) was 4.7-fold faster than with ATP(Mg) alone and was stimulated 2-fold by Cdc42(GTPgammaS). However, gamma-PAK autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS) did not phosphorylate peptide or protein substrates in the presence of ATP(Mn), indicating that gamma-PAK can utilize ATP(Mn) for autophosphorylation but not for phosphorylation of exogenous substrates. Tryptic phosphopeptide maps of gamma-PAK autophosphorylated with ATP(Mg) alone showed 3 phosphopeptides, while with Cdc42(GTPgammaS) a total of 9 major phosphopeptides was observed. When gamma-PAK was autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS), 7 major phosphopeptides were observed, which were identical to peptides obtained with Cdc42(GTPgammaS) and ATP(Mg). Utilizing a recombinant mutant of gamma-PAK with alanine replacing threonine 402 in the catalytic region (T402A), it was determined that the two additional phosphopeptides observed in active PAK (peptides 7 and 8) were due to phosphorylation of threonine 402. These results show that Mn sustains autophosphorylation on serine but does not support autophosphorylation of threonine 402, which is required for activity toward exogenous substrates, or phosphorylation of these substrates.

Cleavage and activation of p21-activated protein kinase gamma-PAK by CPP32 (caspase 3). Effects of autophosphorylation on activity.

p21-activated protein kinase gamma-PAK (Pak2, PAK I) is cleaved by CPP32 (caspase 3) during apoptosis and plays a key role in regulation of cell death. In vitro, CPP32 cleaves recombinant gamma-PAK into two peptides; 1-212 contains the majority of the regulatory domain whereas 213-524 contains 34 amino acids of the regulatory domain plus the entire catalytic domain. Following cleavage, both peptides become autophosphorylated with [gamma-32P]ATP. Peptide 1-212 migrates at 27,000 daltons (p27) upon SDS-polyacrylamide gel electrophoresis and at 32,000 daltons following autophosphorylation on serine (p27P); the catalytic subunit migrates at 34,000 daltons (p34) before and after autophosphorylation on threonine. Following caspase cleavage, a significant lag (approximately 5 min) is observed before autophosphorylation and activity are detected. When gamma-PAK is autophosphorylated with ATP(Mg) alone and then cleaved, only p27 contains phosphate, and the enzyme is inactive with exogenous substrate. After autophosphorylation of gamma-PAK in the presence of Cdc42(GTPgammaS) or histone 4, both cleavage products contain phosphate and gamma-PAK is catalytically active. Mutation of the conserved Thr-402 to alanine greatly reduces autophosphorylation and protein kinase activity following cleavage. Thus activation of gamma-PAK via cleavage by CPP32 is a two-step mechanism wherein autophosphorylation of the regulatory domain is a priming step, and activation coincides with autophosphorylation of the catalytic domain.

The iron-sulfur cluster of iron regulatory protein 1 modulates the accessibility of RNA binding and phosphorylation sites.

Iron regulatory protein 1 (IRP1) modulates iron metabolism by binding to mRNAs encoding proteins involved in the uptake, storage, and metabolic utilization of iron. Iron regulates IRP1 function by promoting assembly of an iron-sulfur cluster in the apo or RNA binding form, thereby converting it to the active holo or cytoplasmic aconitase form. In continuing our studies on phosphoregulation of IRP1 by protein kinase C (PKC), we noted that the purified apoprotein was more efficiently phosphorylated than was the form partially purified from liver cytosol by chromatography on DEAE-Sepharose which had characteristics of the [3Fe-4S] form of the protein. RNA binding measurements revealed a 20-fold increase in RNA binding affinity and a 4-5-fold higher rate of phosphorylation after removal of the Fe-S cluster from the highly purified [4Fe-4S] form. Phosphorylation of apo-IRP1 by PKC was specifically inhibited by IRE-containing RNA. The RNA binding form had a more open structure as judged by its much greater sensitivity to limited cleavage by a number of proteases. N-Terminal sequencing of chymotryptic peptides of apo-IRP1 demonstrated an increased accessibility to proteolysis of sites (residues 132 and 504) near or within the putative cleft of the protein, including regions that are thought to be involved in RNA binding (residues 116-151) and phosphoregulation (Ser 138). Enhanced cleavage was also observed in the proposed hinge linker region (residue 623) on the surface of the protein opposite from the cleft. Taken together, our results indicate that significant structural changes occur in IRP1 during cluster insertion or removal that affect the accessibility to RNA binding and phosphorylation sites.

Determinants for substrate phosphorylation by p21-activated protein kinase (gamma-PAK).

gamma-PAK, originally designated PAK I and subsequently identified as a member of the p21-activated protein kinase family, has been shown to have cytostatic properties and to be involved in maintaining cells in a nondividing state [Rooney, R. D., et al., (1996) J. Biol. Chem. 271, 21498-21504]. The determinants for phosphorylation of substrates by gamma-PAK have been identified by examining the kinetics of phosphorylation of a series of synthetic peptides patterned after the sequence KKRKSGL, which is the site phosphorylated by gamma-PAK in the Rous sarcoma virus nucleocapsid protein NC in vivo and in vitro. With these peptides, the recognition sequence for gamma-PAK has been shown to contain two basic amino acids in the -2 and -3 positions, as represented by (K/R)RXS, in which the -2 position is an arginine, the -3 position is an arginine or a lysine, and X can be an acidic, basic, or neutral amino acid. A basic amino acid in the -1 or -4 position improves the rate of phosphorylation by increasing the Vmax and decreasing the Km. An acidic amino acid in the -1 position increases the rate (2.5-fold), as does an acidic residue in the -4 position, although to a lower extent (1.6-fold). Proline in the -1 or +1 position has a deleterious effect and inhibits phosphorylation by gamma-PAK. The substrate requirements of protein kinases that recognize basic amino acids on the N-terminal side of the phosphorylatable residue such as cAMP-dependent protein kinase (PKA) and Ca2+/phospholipid-dependent protein kinase (PKC) have been compared with gamma-PAK using the same peptides. An acidic residue in the -1 position negatively affects PKA and PKC; thus, peptides containing the sequence KRES can be used to identify gamma-PAK.

Cleavage arrest of early frog embryos by the G protein-activated protein kinase PAK I.

PAK I is a member of the PAK (p21-activated protein kinase) family and is activated by Cdc42 (Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996) J. Biol. Chem. 271, 6206-6211). To examine the effects of PAK I on cleavage arrest, subfemtomole amounts of endogenously active (58 kDa) and inactive (60 kDa) PAK I and a tryptic peptide (37 kDa) containing the active catalytic domain were injected into one blastomere of 2-cell frog embryos. Active PAK I resulted in cleavage arrest in the injected blastomere at mitotic metaphase, whereas the uninjected blastomere progressed through mid- to late cleavage. Injection of other protein kinases at similar concentrations had no effect on cleavage. Endogenous PAK I was highly active in frog oocytes, and antibody to PAK I reacted specifically with protein of 58-60 kDa. PAK I protein was decreased at 60 min post-fertilization, with little or no PAK I protein or activity detectable at 80 min post-fertilization or in 2-cell embryos. At the 4-cell stage PAK I protein increased, but the protein kinase was present primarily as an inactive form. Rac2 and Cdc42, but not Rac 1, were identified in oocytes and throughout early embryo development. Thus, PAK I appears to be a potent cytostatic protein kinase involved in maintaining cells in a non-dividing state. PAK I activity is high in oocytes and appears to be regulated by degradation/synthesis and through autophosphorylation via binding of Cdc42. PAK I may act through regulation of the stress-activated protein kinase signaling pathway and/or by direct regulation of multiple metabolic pathways.

Molecular cloning and sequencing of the cytostatic G protein-activated protein kinase PAK I.

The serine/threonine protein kinase PAK I (p2l-activated protein kinase), a ubiquitous multipotential protein kinase of 58-60 kDa, has been shown to have cytostatic properties. Data from our laboratory show that PAK I is highly active in oocytes and quiescent and serum-starved cells, and injection of active PAK I into one blastomere of two-cell frog embryos inhibits cleavage of the injected blastomere. To clone the cDNA encoding PAK I, purified peptides from rabbit PAK I were sequenced, degenerate oligonucleotides were used to isolate PAK I clones from a rabbit spleen library, and the 5'-terminus was obtained by polymerase chain reaction. The entire cDNA sequence extends over 4471 nucleotides, with an open reading frame for a protein of 524 residues and a 3'-noncoding region of 2826 nucleotides. Clones with the same open reading frame but with 3'-noncoding regions of 1055 and 2478 nucleotides were isolated, suggesting the generation of different transcripts by alternative termination of transcription. The amino acid sequence of PAK I shows high homology to the p2l-activated protein kinases from human placenta and rat brain and to yeast STE20. PAK I is activated by Cdc42(GTP). The PAK enzymes have been proposed to regulate the stress-activated protein kinase (also known as the Jun kinase) signaling pathway (Coso, O. A., Chiariello, M., Yu, J.-C., Teramoto, H., Crespo, P., Xu, N., Miki, T., and Gutkind, J. S. (1995) Cell 81, 1137-1146; Minden, A., Lin, A., Claret, F.-X., Abo, A., and Karin, M. (1995) Cell 81, 1147-1157).

A membrane-bound protein kinase from rabbit reticulocytes is an active form of multipotential S6 kinase.

An active ribosomal protein S6 kinase has been highly purified from the membranes of rabbit reticulocytes by chromatography of the Triton X-100 extract on DEAE-cellulose, SP-Sepharose Fast Flow, and by FPLC on Mono Q and Superose-12. The S6 kinase elutes around 40 000 daltons upon gel filtration on Superose-12 or Sephacryl S-200. It has a subunit molecular weight of 40-43 kDa as determined by protein kinase activity following denaturation/renaturation in SDS-polyacrylamide gels containing S6 peptide. It also phosphorylates translational initiation factors eIF-2 and eIF-4F, glycogen synthase, histone 1, histone 2B, myelin basic protein, but not prolactin, skeletal myosin light chain, histone 4, tubulin, and casein. Apparent Km values have been determined to be 15 microM for ATP, 1.2 microM for S6 and 10 microM for S6 peptide. Two-dimensional tryptic phosphopeptide mapping shows the same sites on S6 are phosphorylated as those identified previously with proteolytically activated multipotential S6 kinase from rabbit reticulocytes, previously denoted as protease activated kinase II. Examination of relative rates of phosphorylation and kinetic constants of synthetic peptides based on previously identified phosphorylation sites, indicates a minimum substrate recognition sequence to be arginine at the n - 3 position. Based on these characteristics, including molecular weight and an expanded substrate specificity, the membrane S6 kinase can be distinguished from the p90 (Type I) and p70 (Type II) S6 kinases, and from protein kinase C and the catalytic subunit of cAMP-dependent protein kinase.

Iron-responsive element-binding protein. Phosphorylation by protein kinase C.

The iron-responsive element-binding protein (IRE-BP) is a cytosolic RNA-binding protein that functions in the maintenance of iron homeostasis by post-transcriptionally regulating transferrin receptor and ferritin synthesis. Little is known concerning how factors other than iron may modulate the activity of this central regulator of cellular iron utilization. We present evidence indicating that phosphorylation of the IRE-BP by protein kinase C (PKC) could provide a mechanism for regulation of IRE-BP function. Purified rat liver IRE-BP was phosphorylated by PKC up to 1.3 mol of phosphate/mol of protein with Ser the modified amino acid. Ser was also the phosphoacceptor in the IRE-BP in intact cells. The Km of PKC for the IRE-BP was 0.4 microM. Tryptic phosphopeptide mapping identified one major phosphopeptide plus several other peptides with lesser amounts of phosphate. Synthetic peptides of the IRE-BP containing Ser 138 (site A) and Ser 711 (site B) were phosphorylated by PKC. In HL 60 cells, addition of phorbol 12-myristate 13-acetate (PMA) stimulated IRE-BP phosphorylation within 30 min and increased high affinity IRE RNA binding activity 2-fold. After 90 min, the level of phosphorylation had increased further, and high affinity IRE RNA binding activity had increased 3-fold above the control. Incorporation of [35S]Met into immunoprecipitable IRE-BP was not altered in cells treated with PMA for 30 or 90 min. PMA also stimulated IRE-BP phosphorylation in rat fibroblasts. Taken together, our studies begin to define a novel mechanism by which hormones, growth factors, and other agents may regulate cellular iron utilization through specific phosphoregulation of the IRE-BP.

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. Grässer, 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.

Modulation of the mitogenic activity of eukaryotic translation initiation factor-4E by protein kinase C.

Eukaryotic initiation factor-4E (eIF-4E) binds to the cap structure of eukaryotic mRNAs and is a component of the cap-binding protein complex eIF-4F. eIF-4E is present in cells in limiting concentrations and is phosphorylated both in vivo and in vitro by protein kinase C (PKC). Recently, eIF-4E has been implicated as an intracellular transducer of extracellular growth signals; microinjection of recombinant eIF-4E into quiescent NIH 3T3 cells induced DNA synthesis. In the present report, the mitogenic activity of eIF-4E was examined after coinjection with PKC. Recombinant eIF-4E was phosphorylated by PKC at the same amino acid that is phosphorylated in cultured cells and reticulocytes in response to phorbol ester. At limiting concentrations of eIF-4E, coinjection with PKC induced a fivefold increase in the mitogenic activity of eIF-4E. Injection of PKC alone or coinjection of eIF-4E with cAMP-dependent protein kinase (PKA) or the Raf protein had no effect. These results suggest that the mitogenic activity of eIF-4E is enhanced by PKC-specific phosphorylation and that phosphate addition is a rate-limiting step in eIF-4E activity.

Characterization of the catalytic subunit of casein kinase II expressed in Escherichia coli and regulation of activity.

The catalytic (alpha) subunit of casein kinase II from Drosophila, cloned and expressed in Escherichia coli (Saxena, A., Padmanabha, R., and Glover, C. V. C., (1987) Mol. Cell. Biol. 7, 3409-3417), has been purified and characterized, and the properties have been compared to those of the holoenzyme. The catalytic subunit exhibits protein kinase activity with casein as substrate and is autophosphorylated. The specific activity of the purified subunit is 6% of the activity of the holoenzyme from reticulocytes or from Drosophila. The alpha subunit is a monomer, eluting at Mr = 40,000 upon gel filtration in high salt, but as part of an aggregate in low salt. The alpha subunit has been purified to apparent homogeneity by sequential chromatography on DEAE-cellulose, Mono S, and Mono Q. A single band, Mr = 37,000, is detected by silver staining following polyacrylamide gel electrophoresis. The isolated alpha subunit displays apparent Km values for beta casein, ATP, and GTP similar to those of the holoenzyme. The activity of the alpha subunit is inhibited by heparin with an I50 of 0.1-0.3 micrograms/ml, a value similar to that observed for the holoenzyme; autophosphorylation is also inhibited by heparin. Polylysine has no stimulatory effect on the activity of the catalytic subunit, as measured with casein and by autophosphorylation, but stimulates both activities with the holoenzyme. When physiological substrates for casein kinase II are examined, glycogen synthase and eukaryotic initiation factor 3 (eIF-3) (p120) are phosphorylated by the alpha subunit at a rate equivalent to that of the holoenzyme, while phosphorylation of eIF-3 (p67) is reduced 9-fold and eIF-2 beta is not modified. From these data, it can be concluded that the alpha subunit of casein kinase II is sufficient for catalysis, is autophosphorylated, and can be directly inhibited by heparin, whereas the beta subunit mediates the effects of basic stimulatory compounds and is involved in recognition and/or binding to specific physiological substrates.

A ribosomal protein S6 kinase copurifies with phosphatase-1 activating factor.

A highly purified preparation of phosphatase-activating kinase (Fa) from rabbit skeletal muscle phosphorylated ribosomal protein S6. The two activities copurified on DEAE-Sephadex, CM-Sephadex, and phosphocellulose chromatography and upon further chromatography on Sephacryl S-300 and FPLC Mono-S and Mono-Q columns. On the latter column, two separate peaks of Fa activity were observed when it was developed in Tris buffer as opposed to beta-glycerophosphate. S6 kinase activity was obtained only with the Fa which adhered to the resin. The Mr of the Fa and S6 activities was determined to be 83,200 by gel permeation on a Sephacryl S-300 column. The Fa preparation phosphorylated serine residues on S6; two tryptic phosphopeptides, A and C, were identified by two-dimensional phosphopeptide analysis. The enzyme also showed good activity toward initiation factor eIF-4B. Based on specificity toward ribosomal proteins and initiation factors, the Fa and a mitogen-stimulated S6 kinase purified from insulin-stimulated 3T3-L1 cells were similar. These results suggest that a form of Fa and an insulin-stimulated S6 kinase may be related or closely associated.

Association of initiation factor eIF-4E in a cap binding protein complex (eIF-4F) is critical for and enhances phosphorylation by protein kinase C.

Phosphorylation by protein kinase C of the mRNA cap binding protein purified as part of a cap binding protein complex (eIF-4F) or as a single protein (eIF-4E), has been examined. Significant phosphorylation (up to 1 mol of phosphate/mol of p25 subunit) occurs only when the protein is part of the eIF-4F complex. With purified eIF-4E, using the same conditions, up to 0.1 mol of phosphate can be incorporated. Tryptic phosphopeptide maps show that the site phosphorylated in the Mr 25,000 subunit of eIF-4F (eIF-4F p25) is the same as that modified in purified eIF-4E. Kinetic measurements obtained from initial rates indicate that the Km values for eIF-4F and eIF-4E are similar, although the Vmax is 5-6 times higher for the complex. Dephosphorylation of eIF-4F p25, previously phosphorylated with protein kinase C, occurs in reticulocyte lysate with a half-life of 15-20 min, whereas little dephosphorylation is observed after 15 min with the purified phosphorylated eIF-4E. Phosphorylation of eIF-4F on the p220 and p25 subunits does not affect the stability of the complex as indicated by gel filtration on Sephacryl S-300. However, addition of non-phosphorylated eIF-4E to the phosphorylated complex results in the dissociation of the complex. These results suggest that interaction of p25 with other subunits in the complex greatly affects phosphorylation/dephosphorylation of p25. Since the rate of phosphorylation/dephosphorylation is significantly greater in the complex, regulation of the cap binding protein by phosphorylation appears to occur primarily on eIF-4F.

Phosphorylation of avian retrovirus matrix protein by Ca2+/phospholipid-dependent protein kinase.

The matrix protein from avian myeloblastosis virus and the Rous sarcoma virus, Prague C strain, is a phosphoprotein. A comparison of the amino acid sequences shows these phosphoproteins are very similar. The sites of phosphorylation of the matrix protein purified from virions are identified as serine residues 68 and 106. Treatment with purified rabbit skeletal-muscle protein phosphatase 1 or 2A, selectively releases phosphate from serine 68, while alkali treatment releases phosphate from both sites. When analyzed as a substrate for six different protein kinases, only the Ca2+/phospholipid-dependent protein kinase modifies the matrix protein. The serine residues phosphorylated in vivo are identical to those phosphorylated in vitro by this protein kinase. The role of these phosphorylation events in viral production is discussed.