Normal cellular and transformation-associated abl proteins share common sites for protein kinase C phosphorylation.

Viral transduction and chromosomal translocations of the c-abl gene result in the synthesis of abl proteins with structurally altered amino termini. These altered forms of the abl protein, but not the c-abl proteins, are detectably phosphorylated on tyrosine in vivo. In contrast, all forms of the abl protein are phosphorylated on serine following in vivo labeling with Pi. Treatment of NIH-3T3 cells with protein kinase C activators resulted in a four- to eightfold increase in the phosphorylation of murine c-abl due to modification of two serines on the c-abl protein. Purified protein kinase C phosphorylated all abl proteins at the same two sites. Both sites are precisely conserved in murine and human abl proteins. The sites on the abl proteins were found near the carboxy terminus. In contrast, for the epidermal growth factor receptor (T. Hunter, N. Ling, and J. A. Cooper, Nature [London] 311:480-483, 1984) and pp60src (K. L. Gould, J. R. Woodgett, J. A. Cooper, J. E. Buss, D. Shalloway, and T. Hunter, Cell 42:849-857, 1985), the sites of protein kinase C phosphorylation are amino-terminal to the kinase domain. The abl carboxy-terminal region is not necessary for the tyrosine kinase activity or transformation potential of the viral abl protein and may represent a regulatory domain. Using an in vitro immune complex kinase assay, we were not able to correlate reproducible changes in c-abl activity with phosphorylation by protein kinase C. However, the high degree of conservation of the phosphorylation sites for protein kinase C between human and mouse abl proteins suggests an important functional role.

Expression of recombinant elongation factor 1 beta from rabbit in Escherichia coli. Phosphorylation by casein kinase II.

The beta subunit of eukaryotic elongation factor 1 (EF-1) catalyzes the GDP/GTP exchange activity on EF-1 alpha. In these studies, two cDNAs for the beta subunit of EF-1 from rabbit are cloned and sequenced. The cDNAs consist of 808 and 798 bp and are identical except for the 5' leader sequences of 67 and 57 bp. Both cDNAs code for a protein of 225 amino acids. Using the pT7-7 expression vector, EF-1 beta was expressed in Escherichia coli and purified to apparent homogeneity by chromatography on DEAE-cellulose and FPLC on Superose 12 and Mono Q. EF-1 beta was highly phosphorylated by casein kinase II, with up to 1.3 mol of phosphate incorporated per mol protein. From microsequence analysis and manual Edman degradation, the majority of the phosphate was shown to be present in serine 106 in the peptide DLFGS106DDEEES112EEA. Serine 112 was also phosphorylated by casein kinase II, but to a lesser extent. Previously, little phosphorylation of the beta subunit by casein kinase II was observed in native EF-1 unless GDP was bound to the alpha subunit (Palen, E., Venema, R.C., Chang, Y-W.E. and Traugh, J.A. (1994) Biochemistry, 8515-8520). In contrast, purified recombinant EF-1 beta was highly and specifically phosphorylated by casein kinase II; GDP and polylysine had little effect on the rate of phosphorylation of the purified subunit.

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.

Protease-activated kinase II as the mediator of epidermal growth factor-stimulated phosphorylation of ribosomal protein S6.

Epidermal growth factor stimulates phosphorylation of ribosomal protein S6 in serum-starved Swiss 3T3 cells, leading to the formation of highly phosphorylated derivatives containing 4-5 phosphates. Two-dimensional analysis of tryptic phosphopeptides of S6 shows an identical pattern to the ones obtained previously in other cells in response to insulin and the tumor promoting phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate. This suggests a common intracellular mediator of S6 phosphorylation by different growth promoting agents. It is proposed that the potential mediator of this phosphorylation is the Ca2+-independent, cAMP-independent protein kinase, protease activated kinase II, as shown by the extent of phosphorylation and the tryptic phosphopeptide maps of S6 with highly purified enzyme [(1983) J. Biol. Chem. 258, 13998-14002].

Identification of three protein kinases which phosphorylate threonyl-tRNA synthetase from rat liver.

Threonyl-tRNA synthetase is phosphorylated in Chinese hamster ovary cells labeled with 32Pi [(1984) J. Biol. Chem. 259, 11160-11161]. Phosphorylation of the purified synthetase from rat liver has been examined with five different protein kinases. Three of the enzymes phosphorylate the synthetase, protease activated kinase I, the cAMP-dependent protein kinase, and the Ca2+, phospholipid-dependent protein kinase. Phosphorylation occurs exclusively on seryl residues. Two-dimensional phosphopeptide maps of tryptic digests of the phosphorylated synthetase are distinct with each protein kinase. These data suggest that multiple phosphorylation of the synthetase may occur in vivo.

Comparison of phosphorylation of elongation factor 1 from different species by casein kinase II.

One subunit of EF-1 or EF-1 beta gamma from Artemia salina, wheat germ and rabbit reticulocytes is modified by casein kinase II. The subunit corresponds to the low Mr subunit of EF-1 (26,000-36,000) which functions along with a higher Mr subunit (46,000-48,000), to catalyze the exchange of GDP for GTP on EF-1 alpha. The factor from Artemia and wheat germ is phosphorylated directly on serine by casein kinase II whereas a modulatory compound is required for phosphorylation of EF-1 from reticulocytes. Polylysine increases the rate of phosphorylation of EF-1 from reticulocytes by 24-fold; both serine and threonine are modified. This suggests that polylysine may be substituting for a physiological regulatory compound which modulates phosphorylation in vivo.

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.

Stimulation of translation in 3T3-L1 cells in response to insulin and phorbol ester is directly correlated with increased phosphate labelling of initiation factor (eIF-) 4F and ribosomal protein S6.

One of the earliest responses to growth-promoting compounds in quiescent or serum-starved cells is stimulation of phosphorylation of ribosomal protein S6. Exposure of 3T3-L1 cells to insulin or phorbol ester (phorbol 12-myristate 13-acetate, PMA) also promotes phosphorylation of specific initiation factors. In this study, stimulation of phosphate labelling of S6, eIF-4F and eIF-4B in response to insulin and PMA has been examined in 3T3-L1 cells and compared with changes in protein synthesis. The rate of phosphate incorporation into eIF-4F and S6 is rapid and parallels the transient stimulation of protein synthesis observed with insulin. Whilst a similar correlation exists with PMA, the response is not as great as with insulin, but is more sustained. A role for the co-ordinate phosphorylation of initiation factors and ribosomal protein S6 in the stimulation of protein synthesis is discussed.

Analysis of the ATP/GTP binding site of casein kinase II by site-directed mutagenesis.

Casein kinase II is one of only a few protein kinases which effectively utilize ATP and GTP in the phosphotransferase reaction. Two residues conserved in the ATP binding domain of other protein kinases are unique to the catalytic (alpha) subunit of casein kinase II. Val-66 is present in subdomain II and Trp-176 in subdomain VII, while > 95% of the other protein kinases contain alanine and phenylalanine, respectively. The residues in the alpha subunit of casein kinase II were changed to the conserved residues via single and double mutations by site-directed mutagenesis. These mutations enhanced the utilization of ATP over GTP by altering the K(m) values of the alpha subunit for ATP and GTP. Following reconstitution of the catalytic subunit with the regulatory (beta) subunit, both the K(m) and Vmax values of the reconstituted alpha 2 beta 2 holoenzyme were altered. Interestingly, the mutations also reduced or eliminated the 4- to 5-fold increase in catalytic activity observed with the holoenzyme over that of the alpha subunit alone. This was due to changes in secondary structure of the holoenzyme as shown by UV circular dichroism spectroscopy. Taken together, the data indicate that utilization of both ATP and GTP can be directly correlated with stimulation of catalytic activity by the regulatory subunit and suggest a co-evolution of these separate functions.