Vasopressin dependent tyrosine phosphorylation of a 38 kDa protein in human platelets.

Arginine vasopressin administration (10(-10)-10(-6) M) to isolated human platelets induces an increase in the specific immunoblotting of a 38 kDa protein revealed by a phosphotyrosine antibody. This signal is biphasic with maximal stimulation within one minute. Neither forskolin (10(-5) M) nor phorbol ester (10(-6) M) produces a similar 38 kDa signal. The specific immunoblotted signals are competitively abolished by 1 mM phosphotyrosine but not phosphoserine or phosphothreonine. Electrophoretic separation at pH 3.5 of the acid hydrolysates of the 38 kDa proteins reveals a vasopressin dependent increase in levels of phosphotyrosine as well as phosphoserine and phosphothreonine. The 38 kDa phosphorylation is also induced by the specific arginine vasopressin V1 receptor agonist (Phe2Orn8Vastocina) and blocked by the V1 receptor antagonist [desGly(NH2)d(CH2)5Tyr(Me) AVPb]. These observations suggest that arginine vasopressin signal transduction may be associated with the tyrosine phosphorylation of a 38 kDa protein.     

Expression of c-myc gene in human ovary carcinoma cells treated with vanadate

The widely accepted hypothesis of vanadate action on cells postulates that this ion inhibits protein phosphatase(s) that dephosphorylates protein phosphotyrosine residues. This inhibition causes tyrosine hyperphosphorylation of cell proteins followed by changes in physiological action of phosphoproteins resulting in stimulation of cell proliferation, expression of protooncogenes, and transient cell transformation. We have found that treatment of human ovary carcinoma (CaOv) cells with vanadate causes the increase in total protein phosphorylation from 1.5- to 2.0-fold whereas the ratio between phosphoserine, phosphothreonine, and phosphotyrosine content remains unchanged. At the same time, enhancement of c-myc gene expression (not c-fos) was observed. Hence, the increase in the ratio of phosphotyrosine to phosphoserine and phosphothreonine is not an obligatory intermediate stage before vanadate-dependent activation of c-myc expression.     

Epidermal growth factor induces rapid tyrosine phosphorylation of proteins in A431 human tumor cells

Addition of EGF to A431 cells at physiological concentrations causes a rapid three- to four-fold increase in the abundance of phosphotyrosine in cellular protein. The increase is essentially complete within 1 min and is maintained for several hours. No change in phosphotyrosine levels is found with fibroblast growth factor or insulin. Two phosphoproteins (molecular weights of 39 and 81 kd) containing phosphotyrosine appear de novo upon administration of EGF to A431 cells. The EGF receptor itself is a phosphoprotein containing phosphotyrosine as well as phosphoserine and phosphothreonine. Changes in the phosphorylation pattern of the EGF receptor are seen upon treatment of A431 cells with EGF. Increased phosphorylation of tyrosine is the most rapid response of cells to EGF known, and may play an important role in the biological effects of EGF.     

A sensitive magnetic bead method for the detection and identification of tyrosine phosphorylation in proteins by MALDI‐TOF/TOF MS

Phosphorylation is one of the most important PTMs and is estimated to occur on 30% of the mammalian proteome. Its perturbed regulation has been implicated in many pathologies. The rarity of phosphotyrosine compared with phosphoserine or phosphothreonine is prompting the development of more sensitive approaches because proteomic technologies that are currently used to assess tyrosine phosphorylation in proteins are inadequate, identifying only a fraction of the predicted tyrosine phosphoproteome. Here we describe the development of a reproducible, high‐sensitivity methodology for the detection and mapping of phosphotyrosine residues by MS. The anti‐phosphotyrosine antibody 4G10 was coupled covalently to super para‐magnetic beads or by affinity to super para‐magnetic beads with protein G covalently attached. Using this approach, we successfully enriched phosphotyrosine peptides mixed with non‐phosphorylated peptides at a ratio of up to 1:200, enabling detection at a level representing the highest sensitivity reported for tyrosine phosphorylation. The beads were subsequently used to enrich tyrosine phosphopeptides from a digest of the in vitro‐phosphorylated recombinant β‐intracellular region of the granulocyte‐macrophage colony‐stimulating factor receptor, which was subsequently analysed by MALDI‐TOF/TOF MS. Our results define this methodology as a sensitive approach for tyrosine phosphoproteome analysis.     

Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity

The regulation of kinase activity associated with insulin receptor by phosphorylation and dephosphorylation has been examined using partially purified receptor immobilized on insulin-agarose. The immobilized receptor preparation exhibits predominately tyrosine but also serine and threonine kinase activities toward insulin receptor beta subunit and exogenous histone. Phosphorylation of the insulin receptor preparation with increasing concentrations of unlabeled ATP, followed by washing to remove the unreacted ATP, results in a progressive activation of the receptor kinase activity when assayed in the presence of histone and [gamma-32P]ATP. A maximal 4-fold activation is achieved by prior incubation of receptor with concentrations of ATP approaching 1 mM. High pressure liquid chromatographic analysis of tryptic hydrolysates of the 32P-labeled insulin receptor beta subunit reveals three domains of phosphorylation (designated peaks 1, 2, and 3). Phosphotyrosine and phosphoserine residues are present in these three domains while peak 2 contains phosphothreonine as well. Thus, at least seven sites are available for phosphorylation on the beta subunit of the insulin receptor. Incubation of the phosphorylated insulin receptor with alkaline phosphatase at 15 degrees C results in the selective dephosphorylation of the phosphotyrosine residues on the beta subunit of the receptor while the phosphoserine and phosphothreonine contents are not affected. The dephosphorylation of the receptor is accompanied by a marked 65% inhibition of the receptor kinase activity. Almost 90% of the decrease in [32P]phosphate content of the receptor after alkaline phosphatase treatment is accounted for by a decrease in phosphotyrosine content in peak 2, while very small decreases are observed in peaks 1 and 3, respectively. These results demonstrate that the extent of phosphorylation of tyrosine residues in receptor domain 2 closely parallels the receptor kinase activity state, suggesting phosphorylation of this domain may play a key role in regulating the insulin receptor tyrosine kinase.     

Protein tyrosine phosphorylation in the cyanobacterium Anabaena sp. strain PCC 7120.

Components of a protein tyrosine phosphorylation/dephosphorylation network were identified in the cyanobacterium Anabaena sp. strain PCC 7120. Three phosphotyrosine (P-Tyr) proteins of 27, 36, and 52 kDa were identified through their conspicuous immunoreactions with RC20H monoclonal antibodies specific for P-Tyr. These immunoreactions were outcompeted completely by free P-Tyr (5 mM) but not by phosphoserine or phosphothreonine. The P-Tyr content of the three major P-Tyr proteins and several minor proteins increased with their time of incubation in the presence of Mg-ATP and the protein phosphatase inhibitors sodium orthovanadate and sodium fluoride. Incubation of the same extracts with [gamma-32P]ATP but not [alpha-32P]ATP led to the phosphorylation of five polypeptides with molecular masses of 20, 27, 52, 85, and 100 kDa. Human placental protein tyrosine phosphatase 1B, with absolute specificity for P-Tyr, liberated significant quantities of 32Pi from four of the polypeptides, confirming that a portion of the protein-bound phosphate was present as 32P-Tyr. Alkaline phosphatase and the dual-specificity protein phosphatase IphP from the cyanobacterium Nostoc commune UTEX 584 also dephosphorylated these proteins and did so with greater apparent efficiency. Two of the polypeptides were partially purified, and phosphoamino analysis identified 32P-Tyr, [32P]phosphoserine, and [32P]phosphothreonine. Anabaena sp. strain PCC 7120 cell extracts contained a protein tyrosine phosphatase activity that was abolished in the presence of sodium orthovanadate and inhibited significantly by the sulfhydryl-modifying agents p-hydroxymercuriphenylsulfonic acid and p-hydroxymercuribenzoate as well as by heparin. In Anabaena sp. strain PCC 7120 the presence and/or phosphorylation status of P-Tyr proteins was influenced by incident photon flux density.     

Protein phosphorylation in purple photosynthetic bacteria

Endogenous protein phosphorylation was shown in both in vitro and in vivo experiments in R. rubrum and in other purple photosynthetic bacteria. Among the substrates of this protein kinase activity the apoproteins of the light harvesting complex were tentatively identified. Phosphoamino acid analysis revealed the presence of phosphoserine, phosphothreonine and phosphotyrosine in R. rubrum. A tyrosine kinase was partially purified in the same bacteria.     

A novel approach for evaluating tyrosine kinase activity based on the radioimmunological determination of phosphotyrosine.

A novel technique was designed to conveniently determine substrate phosphorylation by tyrosine kinase. The technique is based on quantitation of phosphotyrosine content of the phosphoproteins, generated during the enzyme reaction, by radioimmunoassay. Here, we utilized high-titer monoclonal antibodies to phosphotyrosine, and radioiodinated bovine serum albumin-phosphotyrosine conjugate. The radiolabeled antigen was displaced from the complex formed in the assay by unlabeled phosphotyrosine, phosphotyrosine derivatives or phosphotyrosine-containing protein substrates. Half-maximal displacement was achieved at 0.4 +/- 0.05 microM by free phosphotyrosine, and at 40 +/- 3 and 45 +/- 4 nM by acetyl-phosphotyrosine and acetyl-phosphotyrosyl-glycine ethyl ester, respectively. Neither phosphoserine, phosphothreonine nor ATP cross-reacted with the phosphotyrosine antibodies. None of the components of the enzyme reaction interfered in the RIA. The method allows quantitation of the incorporated phosphate into tyrosyl residues without interference of serine/threonine phosphorylation. This technique avoids the use of short-lived [gamma-32P]ATP and omits the separation of the phosphorylated substrate from excess nucleotide.     

Colony stimulating factor-1 (CSF-1) stimulates temperature dependent phosphorylation and activation of the RAF-1 proto-oncogene product.

The serine/threonine kinase RAF-1 is phosphorylated in intact macrophages in response to CSF-1 at 37 degrees C The augmented phosphorylation of RAF-1 and a concomitant increase in RAF-1 associated serine/threonine kinase activity are kinetically later events than CSF-1 induced protein tyrosine phosphorylation. Furthermore, phosphoamino acid analysis of RAF-1 reveals the presence of phosphoserine, trace amounts of phosphothreonine but no phosphotyrosine and the phosphorylated RAF-1 does not react with anti-phosphotyrosine antibodies. In contrast to CSF-1 induced protein tyrosine phosphorylation, RAF-1 phosphorylation and activation are temperature dependent and do not occur at 4 degrees C. Furthermore, coprecipitation experiments failed to reveal any noncovalent association of RAF-1 with the CSF-1 receptor. Thus, while RAF-1 is not a direct substrate for the CSF-1 receptor tyrosine kinase in vivo, its temperature dependent phosphorylation and activation represent an intriguing aspect of the CSF-1 response.     

Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings

A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.     

Vinculin, a cytoskeletal substrate of protein kinase C

Vinculin, a cytoskeletal protein localized at adhesion plaques, is a phosphoprotein containing phosphoserine, phosphothreonine, and phosphotyrosine. Vinculin has been previously shown to be a substrate for pp60src, a phosphotyrosine protein kinase, but the kinase(s) responsible for phosphorylation of the other amino acid residues is unknown. The present report examines the phosphorylation of vinculin by various serine- and threonine-specific protein kinases. Only protein kinase C, the calcium-activated phospholipid-dependent protein kinase, phosphorylates vinculin at a significant rate (24 nmol/min/mg) and displays marked specificity for vinculin. Both calcium and phosphatidylserine were required for vinculin phosphorylation by protein kinase C. In addition, both phorbol 12,13-dibutyrate (10 nM) and phorbol 12-myristate 13-acetate (10 nM) stimulated vinculin phosphorylation by protein kinase C at a limiting calcium concentration (10(-6) M). Tryptic peptide analysis revealed two major sites of phosphorylation. One site contained phosphoserine and the other contained phosphothreonine. When compared with tryptic maps of vinculin phosphorylated by src kinase, no overlapping phosphorylated peptides were found. The present findings coupled with the plasma membrane location of both these proteins suggest that vinculin may be a physiologic substrate for protein kinase C.     

Acidic and basic fibroblast growth factors stimulate tyrosine kinase activity in vivo

We assessed the ability of acidic and basic fibroblast growth factor (FGF) to stimulate tyrosine kinase activity in intact cells. Immunoblot with polyclonal antiphosphotyrosine antibodies detected a 90-kDa phosphotyrosine-bearing protein in lysates of Swiss 3T3 cells exposed to pituitary-derived FGF, recombinant acidic FGF, or recombinant basic FGF, but not from unstimulated cells or cells exposed to epidermal growth factor or platelet-derived growth factor. Phosphotyrosine and its analogue phenyl phosphate, but not phosphoserine, phosphothreonine, or tyrosine itself, blocked recognition of the 90-kDa protein by antiphosphotyrosine antiserum. A monoclonal antiphosphotyrosine antibody also recognized the 90-kDa protein and was used to partially purify the protein by immunoaffinity chromatography. Phosphoamino acid analysis of the 90 kDa band revealed that it contained 20% phosphotyrosine, 35% phosphothreonine, and 45% phosphoserine. Tyrosine phosphorylation of the 90-kDa protein was detectable within 30 s and reached a plateau within 10 min of FGF addition. The addition of suramin, which blocks the interaction of FGF with its receptor, caused rapid disappearance of the 90 kDa band. Cell fractionation experiments were consistent with the 90-kDa protein being membrane-associated, but cross-linking studies revealed that the FGF receptor had an Mr between 145 and 210 kDa in Swiss 3T3 cells, distinct from the 90-kDa major substrate for tyrosine phosphorylation. These data demonstrate that both acidic and basic FGF activate a tyrosine kinase in vivo leading to phosphorylation of a unique 90-kDa substrate, and they suggest that protein modification by phosphorylation at tyrosine is involved in eliciting the mitogenic effect of FGF.     

Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

Protein phosphorylation was studied in primary cultures of thyroid epithelial cells after the addition of different mitogens: thyrotropin (TSH) acting through cyclic AMP, epidermal growth factor (EGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA). EGF or TPA increased the phosphorylation of five common polypeptides. Among these, two 42-kilodalton proteins contained phosphotyrosine and phosphoserine with or without phosphothreonine. Their characteristics suggested that they are similar to the two 42-kilodalton target proteins for tyrosine protein phosphorylation demonstrated in fibroblasts in response to mitogens. No common phosphorylated proteins were detected in TSH-treated cells and in EGF- or TPA-treated cells. The differences in the protein phosphorylation patterns in response to TSH, EGF, and TPA suggested that the newly emerging cyclic AMP-mediated mitogenic pathway is distinct from the better known growth factor- and tumor promoter-induced pathways.     

Light-Dependent Tyrosine Phosphorylation in the Cyanobacterium Prochlorothrix hollandica

A light-dependent tyrosine kinase activity is present in soluble extracts from the cyanobacterium Prochlorothrix hollandica. The substrate of this tyrosine kinase activity is a soluble 88-kD protein that is phosphorylated when cultures of P. hollandica are adapted to high-light conditions. This phosphoprotein was identified by probing western blots of 32P-labeled soluble proteins from P. hollandica with an antibody specific for phosphotyrosine. This specificity was confirmed by competition experiments in which the antibody binding was abolished completely in the presence of excess phosphotyrosine but not phosphoserine and phosphothreonine. The kinetics of phosphorylation in vivo were determined by probing western blots with this antibody. Within 1 h following a switch from extended darkness to high light (200 [mu]mol photons m-2 s-1), the 88-kD protein was detectable upon India ink staining of western blots. After 3 h, the antibody recognized the phosphorylated form of this polypeptide. Within 6 h of a downshift from high to low light, the 88-kD protein was dephosphorylated. In vitro phosphorylation studies also showed that cell extracts can phosphorylate a tyrosine-containing artificial substrate; acid hydrolysis of both the artificial substrate and the 88-kD protein showed that phosphorylation occurred exclusively on tyrosine residues. Finally, experiments with high-light-adapted Synechococcus sp. PCC7942 suggest that a similar tyrosine phosphorylation event occurs in a phycobilisome-containing cyanobacterium.     

Tumor promoter and epidermal growth factor stimulate phosphorylation of the c-erbB-2 gene product in MKN-7 human adenocarcinoma cells.

Treatment of human adenocarcinoma MKN-7 cells with epidermal growth factor (EGF) or phorbol tetradecanoate acetate (TPA) stimulated phosphorylation of the c-erbB-2 gene product. EGF induced a rapid increase in phosphotyrosine followed by relatively gradual increases in phosphoserine and phosphothreonine. On the other hand, the TPA-induced increase in phosphorylations occurred exclusively on serine and threonine residues. Tryptic phosphopeptide mapping analysis suggested that treatments with EGF and TPA induced phosphorylation of many common sites in the c-erbB-2 gene product. However, in contrast to TPA, EGF increased the phosphorylation of the c-erbB-2 protein in cells whose protein kinase C had been down-regulated by long-term pretreatment with TPA, suggesting that EGF and TPA induce phosphorylation by different mechanisms. Since the c-erbB-2 gene product did not show detectable EGF-binding activity, phosphorylation of tyrosine of the c-erbB-2 gene product might be catalyzed directly by the EGF receptor kinase that was activated by EGF.     

Comparison of three quantitative phosphoproteomic strategies to study receptor tyrosine kinase signaling

There are three quantitative phosphoproteomic strategies most commonly used to study receptor tyrosine kinase (RTK) signaling. These strategies quantify changes in: (1) all three forms of phosphosites (phosphoserine, phosphothreonine and phosphotyrosine) following enrichment of phosphopeptides by titanium dioxide or immobilized metal affinity chromatography; (2) phosphotyrosine sites following anti- phosphotyrosine antibody enrichment of phosphotyrosine peptides; or (3) phosphotyrosine proteins and their binding partners following anti-phosphotyrosine protein immunoprecipitation. However, it is not clear from literature which strategy is more effective. In this study, we assessed the utility of these three phosphoproteomic strategies in RTK signaling studies by using EphB receptor signaling as an example. We used all three strategies with stable isotope labeling with amino acids in cell culture (SILAC) to compare changes in phosphoproteomes upon EphB receptor activation. We used bioinformatic analysis to compare results from the three analyses. Our results show that the three strategies provide complementary information about RTK pathways.     

Large‐scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome‐wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high‐accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high‐confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine‐specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho‐residues in Arabidopsis is similar to that in humans, where over 90 tyrosine‐specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.     

Src kinase regulation by phosphorylation and dephosphorylation

Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPalpha, PTPepsilon, and PTPlambda. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.     

Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40.

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been analyzed by partial proteolysis peptide mapping and phosphoamino acid analysis of the resulting products. At least four sites were found to be phosphorylated. An amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the proline-rich carboxy-terminal end of the molecule, either at position 701 or at position 708. The mutant dl 1265, which is defective in adenovirus helper function, lacked this phosphorylation site. In addition, the carboxy-terminal part of the molecule contained phosphoserine at a more central position. T-antigen-associated proteins of SV40-transformed cell (nonviral T; 51,000 to 55,000 daltons) also contained multiple phosphorylation sites involving at least two serine residues in mouse antigens and an additional threonine residue in rat, human, and monkey antigens. The latter residue and at least one phosphoserine residue were located near one terminus of the human NVT molecule. We did not find any evidence for phosphorylation of tyrosine residues in any of the multiple species of either large T or nonviral T molecules. Several forms of large T antigens were extracted from both SV40-transformed and SV40-infected permissive and nonpermissive cells, and their phosphorylation patterns were compared. No evidence was found for a different phosphorylation pattern of T antigen in transformed cells.     

Cdc25 regulates the phosphorylation and activity of the Xenopus cdk2 protein kinase complex.

The Xenopus cdk2 gene encodes a 32-kDa protein kinase with sequence similarity to the 34-kDa product of the cdc2 gene. Previous studies have shown that the kinase activity of the protein product of the cdk2 gene oscillates in the Xenopus embryonic cell cycle with a high in M-phase and a low in interphase. In the present study cdk2 was found not to be associated with any newly synthesized proteins during the cell cycle, but the enzyme did undergo periodic changes in phosphorylation. Upon exit from metaphase, cdk2 became increasingly phosphorylated on both tyrosine and serine residues, and labeling on these residues increased progressively until entry into mitosis, when tyrosine residues were markedly dephosphorylated. Phosphopeptide mapping of cdk2 demonstrated the major sites of phosphorylation were in a phosphopeptide with a pI of 3.7 that contained both phosphoserine and phosphotyrosine. This phosphopeptide accumulated in egg extracts blocked in S-phase with aphidicolin and was not evident in cdc2 immunoprecipitated under the same conditions. Under the same conditions cdc2 was phosphorylated primarily on a phosphopeptide containing both phosphothreonine and phosphotyrosine residues, most likely threonine 14 and tyrosine 15. Affinity-purified human GST-cdc25 was able to dephosphorylate and activate cdk2 isolated from interphase cells. Phosphopeptide mapping demonstrated that the phosphate was specifically removed from the same phosphopeptide identified as the major in vivo site of phosphorylation. These results demonstrate that cdk2 is regulated in the cell cycle by phosphorylation and dephosphorylation on both serine and tyrosine residues. Moreover, the increased phosphorylation of cdk2 in aphidicolin-blocked extracts and the ability of cdc25 to mediate cdk2 dephosphorylation in vitro suggest the possibility that cdk2 is part of the mechanism ensuring mitosis is not initiated until completion of DNA replication. It also implies cdc25 may have other functions in addition to the regulation of cdc2 kinase activity.