Plk1-mediated stabilization of 53BP1 through USP7 regulates centrosome positioning to maintain bipolarity.

Although 53BP1 has been established well as a mediator in DNA damage response, its function in mitosis is not clearly understood. We found that 53BP1 is a mitotic-binding partner of the kinases Plk1 and AuroraA, and that the binding with Plk1 increases the stability of 53BP1 by accelerating its interaction with the deubiquitinase USP7. Depletion of 53BP1 induces mitotic defects such as chromosomal missegregation, misorientation of spindle poles and the generation of extra centrosomes, which is similar phenotype to USP7-knockdown cells. In addition, 53BP1 depletion reduces the levels of p53 and centromere protein F (CENPF), interacting proteins of 53BP1. These phenotypes induced by 53BP1 depletion were rescued by expression of wild-type or phosphomimic mutant 53BP1 but not by expression of a dephosphomimic mutant. We propose that phosphorylation of 53BP1 at S380 accelerates complex formation with USP7 and CENPF to regulate their stability, thus having a crucial role in proper centrosome positioning, chromosomal alignment, and centrosome number.

Plk1 depletion in nontransformed diploid cells activates the DNA-damage checkpoint.

We previously reported that polo-like kinase 1 (Plk1) depletion by lentivirus-based RNA interference led to mitotic arrest and apoptosis in cancer cells, whereas normal diploid cell lines, hTERT-RPE1 and MCF10A, survived a similar level of depletion. To study homogeneous cell lines, we generated several Plk1-depleted hTERT-RPE1 and MCF10A clones that were derived from single cells depleted of Plk1. We found that in the long term, Plk1 depletion slowed proliferation of hTERT-RPE1 cells, apparently due to attenuated progression through S phase. These cells had altered morphology and were elongated compared with control. In contrast, MCF10A clones with mild levels of depletion showed no obvious phenotype. They appeared to have normal proliferation rates with no cell-cycle arrest. However, one MCF10A clone, which was severely depleted of Plk1, although viable, showed sporadic G2/M arrest and apoptosis. This MCF10A clone and all the hTERT-RPE1 clones displayed evidence of DNA-damage checkpoint activation. These data further support the interpretation that cancer cell lines have a much greater requirement for Plk1 than normal nontransformed diploid cells.

Peripheral Golgi protein GRASP65 is a target of mitotic polo-like kinase (Plk) and Cdc2.

Cell division is characterized by orchestrated events of chromosome segregation, distribution of cellular organelles, and the eventual partitioning and separation of the two daughter cells. Mitotic kinases, including polo-like kinases (Plk), influence multiple events in mitosis. In yeast two-hybrid screens using mammalian Plk C-terminal domain baits, we have identified Golgi peripheral protein GRASP65 (Golgi reassembly stacking protein of 65 kDa) as a Plk-binding protein. GRASP65 appears to function in the postmitotic reassembly of Golgi stacks. In this report we demonstrate binding between Plk and GRASP65 and provide in vitro and in vivo evidence that Plk is a GRASP65 kinase. Moreover, we show that Cdc2 can also phosphorylate GRASP65. In addition, we present data which support the observation that the conserved C terminus of Plk is important for its function. Deletion or frameshift mutations in the conserved C-terminal domain of Plk greatly diminish its ability to phosphorylate GRASP65. These and previous findings suggest that phosphorylation of Golgi components by mitotic kinases may regulate mechanisms of Golgi inheritance during cell division.

Transfection of constitutively active mitogen-activated protein/extracellular signal-regulated kinase kinase confers tumorigenic and metastatic potentials to NIH3T3 cells.

Cellular growth and differentiation are controlled by multiple extracellular signals, many of which activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinases. Components of the MAP kinase pathways also cause oncogenic transformation in their constitutively active forms. Moreover, expression of activated ras can confer metastatic potential upon some cells. Activation of MAP kinases requires phosphorylation of both Thr and Tyr in the catalytic domain by a family of dual-specificity kinases, called MEKs (MAP kinase/ERK kinase). MEK1 is activated by phosphorylation at Ser218 and Ser222 by Raf. Mutation of these two sites to acidic residues, specifically [Asp218], [Asp218, Asp222], and [Glu218, Glu222], results in constitutively active MEK1. Using these mutant variants of MEK1, we showed previously that transfection of NIH/3T3 or Swiss 3T3 cells causes morphological transformation and increases growth on soft agar, independent of ERK activity. The transformed cell lines show increased expression of matrix metalloproteinases 2 and 9 and cathepsin L, proteinases that have been implicated in the metastatic process. We tested NIH3T3 cells transfected with the [Asp218] or [Asp218, Asp222] for metastatic potential after i.v. injection into athymic mice. Parental 3T3 cells formed no tumors grossly or histologically. However, all MEK1 mutant transformants formed macroscopic metastases. Thus, like activated Ras, MEK1 can confer both tumorigenic and metastatic potential upon NIH3T3 cells. These results refine the mechanism through which ras could confer tumorigenic and metastatic potential (ie., the critical determinants of tumorigenic and metastatic potential are downstream of MEK1).

Polo-like kinase 1 as a target for human cytomegalovirus pp65 lower matrix protein.

Human cytomegalovirus (HCMV) pp65 protein is the major constituent of viral dense bodies but is dispensable for viral growth in vitro. pp65 copurifies with a S/T kinase activity and has been implicated in phosphorylation of HCMV IE1 immediate-early protein and its escape from major histocompatibility complex 1 presentation. Furthermore, the presence of pp65 correlates with a virion-associated kinase activity. To clarify the role of pp65, yeast two-hybrid system (THS) screening was performed to identify pp65 cellular partners. A total of 18 out of 48 yeast clones harboring cDNAs for putative pp65 binding proteins encoded the Polo-like kinase 1 (Plk1) C-terminal domain. Plk1 behaved as a bona fide pp65 partner in THS control crosses, and the interaction was confirmed by in vitro binding experiments. Endogenous Plk1 was coimmunoprecipitated with pp65 from transiently transfected COS7 cells. In infected fibroblasts, Plk1 was coimmunoprecipitated with pp65 at late infection stages. Furthermore, Plk1 was detected within wild-type HCMV particles but not within the particles of a pp65-negative mutant (RVAd65). The hydrophilic region of pp65 was phosphorylated in vitro by Plk1. These results suggest that one function of pp65 may be to capture a cell kinase, perhaps in order to alter its activity, nucleotide preference, substrate specificity, or subcellular localization to the advantage of HCMV.

Mutation of the polo-box disrupts localization and mitotic functions of the mammalian polo kinase Plk.

Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic domain, termed the polo-box. The function of the polo-box is currently undefined. The mammalian polo-like kinase Plk is a functional homologue of Saccharomyces cerevisiae Cdc5. Here, we show that Plk localizes at the spindle poles and cytokinetic neck filaments. Without impairing kinase activity, a conservative mutation in the polo-box disrupts the capacity of Plk to complement the defect associated with a cdc5-1 temperature-sensitive mutation and to localize to these subcellular structures. Our data provide evidence that the polo-box plays a critical role in Plk function, likely by directing its subcellular localization.

An analysis of Mek1 signaling in cell proliferation and transformation.

The Mek1 dual specificity protein kinase phosphorylates and activates the mitogen-activated protein kinases Erk1 and Erk2 in response to mitogenic stimulation. The molecular events downstream of Mek and Erk necessary to promote cell cycle entry are largely undefined. In order to study signals emanating from Mek independent of upstream proteins capable of activating multiple signaling pathways, we fused the hormone-binding domain of the estrogen receptor (ER) to the C terminus of constitutively activated Mek1 phosphorylation site mutants. Although 4-OH-tamoxifen stimulation of NIH-3T3 cells expressing constitutively activated Mek-ER resulted in only a small increase in specific activity of the fusion protein, a 5-10 fold increase in total cellular Mek activity was observed over a period of 1-2 days due to an accumulation of fusion protein. Induction of constitutively activated Mek-ER in NIH-3T3 cells resulted in accelerated S phase entry, proliferation in low serum, morphological transformation, and anchorage independent growth. Endogenous Erk1 and Erk2 were phosphorylated with kinetics similar to the elevation of Mek-ER activity. However, elevated Mek-ER activity attenuated subsequent stimulation of Erk1 and Erk2 by serum. 4-OH-tamoxifen stimulation of Mek-ER-expressing fibroblasts also resulted in up-regulation of cyclin D1 expression and down-regulation of p27(Kip1) expression, establishing a direct link between Mek1 and the cell cycle machinery.

Plk is a functional homolog of Saccharomyces cerevisiae Cdc5, and elevated Plk activity induces multiple septation structures.

Plk is a mammalian serine/threonine protein kinase whose activity peaks at the onset of M phase. It is closely related to other mammalian kinases, Snk, Fnk, and Prk, as well as to Xenopus laevis Plx1, Drosophila melanogaster polo, Schizosaccharomyces pombe Plo1, and Saccharomyces cerevisiae Cdc5. The M phase of the cell cycle is a highly coordinated process which insures the equipartition of genetic and cellular materials during cell division. To enable understanding of the function of Plk during M phase progression, various Plk mutants were generated and expressed in Sf9 cells and budding yeast. In vitro kinase assays with Plk immunoprecipitates prepared from Sf9 cells indicate that Glu206 and Thr210 play equally important roles for Plk activity and that replacement of Thr210 with a negatively charged residue elevates Plk specific activity. Ectopic expression of wild-type Plk (Plk WT) complements the cell division defect associated with the cdc5-1 mutation in S. cerevisiae. The degree of complementation correlates closely with the Plk activity measured in vitro, as it is enhanced by a mutationally activated Plk, T210D, but is not observed with the inactive forms K82M, D194N, and D194R. In a CDC5 wild-type background, expression of Plk WT or T210D, but not of inactive forms, induced a sharp accumulation of cells in G1. Consistent with elevated Plk activity, this phenomenon was enhanced by the C-terminally deleted forms WT deltaC and T210D deltaC. Expression of T210D also induced a class of cells with unusually elongated buds which developed multiple septal structures. This was not observed with the C-terminally deleted form T210D deltaC, however. It appears that the C terminus of Plk is not required for the observed cell cycle influence but may be important for polarized cell growth and septal structure formation.

Differential expression of MEK1 and MEK2 during mouse development.

Map/Erk kinase 1 (MEK1) and MEK2 activate the Erk/ MAP kinases and have been implicated in cell growth and differentiation. To investigate the role of MEKs during mouse development, we have examined their expression and activity in various murine tissues during embryonic development and in the adult mouse. MEK2 RNA message is expressed at high levels in all embryonic tissues examined, including all neural tissues, and liver. This can be observed by in situ hybridization of tissue sections of 14.5-day-old mouse embryos, as well as by Northern blot analyses. MEK1, on the other hand, is expressed at very low levels in most embryonic murine tissue but can be detected in developing skeletal muscle. It is expressed at higher levels in adult tissue, particularly in brain, where it is expressed at high levels. Western blot analyses of MEK1 and MEK2 in 14.5-day-old embryonic and adult mouse tissue confirm the RNA analysis. Levels of MEK1 kinase activity are particularly high in adult brain tissues as well. These findings suggest that MEK2 may be the primary Erk/MAP kinase activator during development and that MEK1 may play a role in the proliferative or mitogenic response in adult mouse tissues. This study also raises the possibility that MEK1 and MEK2 might not have redundant functions in cells but may possess unique specificity in their interactions with upstream activators or downstream targets.

Mek1 phosphorylation site mutants activate Raf-1 in NIH 3T3 cells.

MAP (mitogen-activated protein) kinases are activated by a family of dual specificity kinases called Meks (MAP kinase/Erk kinase). Mek1 can be activated by Raf by phosphorylation on serine 218 and serine 222. Mutation of these sites to acidic residues leads to constitutively active Mek1 in some cases. When fibroblast lines were infected with high titer retroviral stocks carrying these Mek1 genes, the resultant transformation and morphological changes correlated with the kinase activity of the respective Mek1 enzymes. Although [Asp218]- and [Asp218,Asp222]Mek immunoprecipitated from clonal cell lines could phosphorylate kinase-inactive Erk1 equally well in vitro, the endogenous MAP kinase activity was 5-7-fold greater in [Asp218]Mek1-infected clonal lines, and did not correlate with the degree of transformation. Analysis of the Erk1 pathway revealed Raf-1 activation, which correlated qualitatively with the MAP kinase activity seen in the [Asp218]- and [Asp218,Asp222]Mek1-infected clonal cell lines. Expression of dominant negative Ras did not affect the elevated Raf-1 activity observed in these cells, however. These data suggest that Mek1 phosphorylation site mutants activate Raf-1 and MAP kinase by a Ras-independent pathway and that the mechanism by which transformation occurs may utilize pathways that are MAP kinase-independent.

A Drosophila gene structurally and functionally homologous to the mammalian 70-kDa s6 kinase gene.

cDNAs encoding the Drosophila 70-kDa S6 kinase (S6K) were isolated by low-stringency hybridization with mammalian p70S6k probes. Conceptual translation of S6k cDNA sequences yields a product containing all of the canonical features typical of serine/threonine kinases and has 78% amino acid identity in the catalytic domain with the human p70S6k homologue. The S6k gene, located at polytene chromosome site 65D, gives rise to two predominant transcripts of 3.0 and 5.0 kb and at least two smaller transcripts (< 3.0 kb) that are found in whole-animal RNAs at all stages of development. Blood cells derived from the hematopoietic organs of ribosomal protein S6 (RpS6air8) mutant animals express higher levels of the smaller S6k transcripts, suggesting tissue- or genotype-specific differences in the regulation of the S6k gene. Drosophila S6K expressed in COS or NIH 3T3 cells phosphorylates mammalian RPS6 in a mitogen-dependent wortmannin- and rapamycin-sensitive manner, suggesting that its regulation is similiar to mammalian p70S6k.

Plk is an M-phase-specific protein kinase and interacts with a kinesin-like protein, CHO1/MKLP-1.

PLK (STPK13) encodes a murine protein kinase closely related to those encoded by the Drosophila melanogaster polo gene and the Saccharomyces cerevisiae CDC5 gene, which are required for normal mitotic and meiotic divisions. Affinity-purified antibody generated against the C-terminal 13 amino acids of Plk specifically recognizes a single polypeptide of 66 kDa in MELC, NIH 3T3, and HeLa cellular extracts. The expression levels of both poly(A)+ PLK mRNA and its encoded protein are most abundant about 17 h after serum stimulation of NIH 3T3 cells. Plk protein begins to accumulate at the S/G2 boundary and reaches the maximum level at the G2/M boundary in continuously cycling cells. Concurrent with cyclin B-associated cdc2 kinase activity, Plk kinase activity sharply peaks at the onset of mitosis. Plk enzymatic activity gradually decreases as M phase proceeds but persists longer than cyclin B-associated cdc2 kinase activity. Plk is localized to the area surrounding the chromosomes in prometaphase, appears condensed as several discrete bands along the spindle axis at the interzone in anaphase, and finally concentrates at the midbody during telophase and cytokinesis. Plk and CHO1/mitotic kinesin-like protein 1 (MKLP-1), which induces microtubule bundling and antiparallel movement in vitro, are colocalized during late M phase. In addition, CHO1/MKLP-1 appears to interact with Plk in vivo and to be phosphorylated by Plk-associated kinase activity in vitro.

Biochemical and biological analysis of Mek1 phosphorylation site mutants.

Recently, we described the constitutive activation of Mek1 by mutation of its two serine phosphorylation sites. We have now characterized the biochemical properties of these Mek1 mutants and performed microinjection experiments to investigate the effect of an activated Mek on oocyte maturation. Single acidic substitution of either serine 218 or 222 activated Mek1 by 10-50 fold. The double acidic substitutions, [Asp218, Asp222] and [Asp218, Glu222], activated Mek1 over 6000-fold. The specific activity of the [Asp218, Asp222] and [Asp218, Glu222] Mek1 mutants, 29 nanomole phosphate per minute per milligram, is similar to that of wild-type Mek1 activated by Raf-1 in vitro. Although the mutants with double acidic substitutions could not be further activated by Raf-1, three of those with single acidic substitution were activated by Raf-1 to the specific activity of activated wild-type Mek1. Injection of the [Asp218, Asp222] Mek1 mutant into Xenopus oocytes activated both MAP kinase and histone H1 kinase and induced germinal vesicle breakdown, an effect that was only partially blocked by inhibition of protein synthesis. These data provide a measure of Mek's potential to influence cell functions and a quantitative basis to assess the biological effects of Mek1 mutants in a variety of circumstances.

Cloning of a human insulin-stimulated protein kinase (ISPK-1) gene and analysis of coding regions and mRNA levels of the ISPK-1 and the protein phosphatase-1 genes in muscle from NIDDM patients.

Complementary DNA encoding three catalytic subunits of protein phosphatase 1 (PP1 alpha, PP1 beta, and PP1 gamma) and the insulin-stimulated protein kinase 1 (ISPK-1) was analyzed for variations in the coding regions related to insulin-resistant glycogen synthesis in skeletal muscle of 30 patients with non-insulin-dependent diabetes mellitus (NIDDM). The human ISPK-1 cDNA was cloned from T-cell leukemia and placental cDNA libraries and mapped to the short arm of the human X chromosome. Single-strand conformation polymorphism (SSCP) analysis identified a total of six variations in the coding regions of the PP1 genes: two in PP1 alpha at codons 90 and 255; one in PP1 beta at codon 67; and three in PP1 gamma at codons 11,269, and 273, respectively. All were, however, silent single nucleotide substitutions. SSCP analysis of the ISPK-1 gene identified one silent polymorphism at codon 266 and one amino acid variant at codon 38 (Ile-->Ser). This variant was primarily found in one male NIDDM patient. This subject, however, did not exhibit an impairment of muscle insulin-stimulated glycogen synthase activation. No significant differences were found in mRNA levels in muscle of the four genes between 15 NIDDM patients and 14 healthy subjects. Our findings suggest that 1) genetic abnormalities in the coding regions of PP1 alpha, PP1 beta, PP1 gamma, and ISPK-1 are unlikely to be frequently occurring causes of the reduced insulin-stimulated activation of the glycogen synthesis in muscle from the analyzed group of NIDDM patients; 2) the mRNA levels of PP1 alpha, PP1 beta, PP1 gamma, and ISPK-1 are normal in muscle from the NIDDM patients; and 3) putative inherited defects in insulin-stimulated activation of muscle glycogen synthesis in patients with insulin-resistant NIDDM may be located further upstream of ISPK-1 in the insulin action cascade.

Negative regulation of mitosis in fission yeast by catalytically inactive pyp1 and pyp2 mutants.

The Schizosaccharomyces pombe genes pyp1+ and pyp2+ encode protein tyrosine phosphatases (PTPases) that act as negative regulators of mitosis upstream of the wee1+/mik1+ pathway. Here we provide evidence that pyp1+ and pyp2+ function independently of cdr1+(nim1+) in the inhibition of mitosis and that the wee1 kinase is not a direct substrate of either PTPase. In a pyp1::ura4 cdc25-22 genetic background, overexpression of either the N-terminal domain of pyp1+ or a catalytically inactive mutant, pyp1C470S, causes cell cycle arrest. This phenotype reverses the suppression of a cdc25 temperature-sensitive mutation at 35 degrees C caused by a pyp1 disruption. Furthermore, pyp1C470S and a catalytically inactive mutant of pyp2, pyp2C630S, induce mitotic delay as do their wild-type counterparts. Analysis of pyp1+ and pyp2+ further reveals that in vitro PTPase activity of pyp1 and pyp2, as well as their biological activity, is dependent on the presence of N-terminal sequences that are not normally considered part of PTPase catalytic domains.

Constitutive activation of Mek1 by mutation of serine phosphorylation sites.

A variety of extracellular signals lead to the phosphorylation and activation of mitogen-activated protein kinases (MAP kinases). An activator of MAP kinases, Mek1, phosphorylates MAP kinases at threonine and tyrosine residues and is itself phosphorylated at serine-218 and -222 by the protooncogene product Raf-1. By introducing negatively charged residues that may mimic the effect of phosphorylation at positions 218 and 222, we have activated the capacity of Mek1 to phosphorylate MAP kinase by > 100-fold. The most effective activation by a single substitution resulted from the introduction of aspartate at position 218, whereas the introduction of either aspartate or glutamate at position 222 was ineffective. Expression of the activated Mek1 phosphorylation-site mutants in COS-7 cells led to the activation of MAP kinase in the cells and resulted in an increase in the mass of the transfected COS-7 cell population, suggesting an important role of Mek1 in the transduction of mitogenic signals.

Raf-1 forms a stable complex with Mek1 and activates Mek1 by serine phosphorylation.

Recombinant Mek1 and Raf-1 proteins produced in Sf9 cells undergo a tight association both in vivo and in vitro, which apparently does not depend on additional factors or the kinase activity of Mek1 or Raf-1. The complex can be disrupted by two polyclonal antibodies raised against Raf-1 peptides. Coinfection with Raf-1 activates Mek1 > 150-fold, and coinfection with Raf-1 and Mek1 activates Erk1 approximately 90-fold. The activation of Mek1 by Raf-1 involves only serine phosphorylation, which is directly proportional to the extent of Mek1 activation. Phosphopeptide maps suggest a single Raf-1 phosphorylation site on mek1.

MEK2 is a kinase related to MEK1 and is differentially expressed in murine tissues.

MEK1 is a dual specificity kinase that phosphorylates and activates the Erk/MAP kinases Erk-1 and Erk-2 by phosphorylating them on threonine and tyrosine. We report the cloning of a second MEK-like complementary DNA, Mek2, which predicts a protein of a molecular weight of 44,500. The MEK2 protein bears substantial sequence homology to MEK1, except at its amino terminus, and at a proline-rich region insert between the conserved kinase subdomains 9 and 10. MEK1 and MEK2 are shown to be encoded by different genes and are located on murine chromosomes 9 and 10, respectively. Northern analysis indicates that Mek2 is expressed at low levels in adult mouse brain and heart tissue, and at higher levels in other tissues examined. Low expression levels of Mek2 in brain tissue are in contrast to the high levels of Mek1 expressed in brain. Mek2 is expressed at high levels in neonatal brain, however. Recombinant MEK2 produced in bacteria phosphorylates a kinase-inactive Erk-1 on tyrosine and threonine, whereas a kinase-inactive mutant MEK2 does not. These findings suggest that MEK2 is a member of a multigene family.

Reconstitution of the Raf-1-MEK-ERK signal transduction pathway in vitro.

Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p21ras can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitope-tagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-1 purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.

Protein tyrosine phosphatase 1B undergoes mitosis-specific phosphorylation on serine.

We have investigated the regulation of protein tyrosine phosphatase 1B (PTP1B) through the cell cycle of HeLa cells. PTP1B from HeLa cells arrested in mitosis migrated more slowly during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than did PTP1B from unsynchronized HeLa cells. To explore whether this mobility shift was caused by phosphorylation, PTP1B was immunoprecipitated from 32Pi-labeled unsynchronized and mitotic HeLa cells. PTP1B from mitotic cells incorporated significantly more 32Pi than did PTP1B from unsynchronized cells. Alkaline phosphatase treatment of mitotic HeLa cell lysates resulted in the conversion of PTP1B to its more rapidly migrating form, confirming that the mobility shift was a result of the mitotic phosphorylation. Phosphoamino acid analysis of PTP1B from mitotic cells revealed that PTP1B became phosphorylated on serine. Dephosphorylation of PTP1B occurred following the release of cells from nocodazole synchronization and was independent of new protein synthesis. This dephosphorylation was inhibited by okadaic acid, a potent inhibitor of types 1 and 2A serine/threonine phosphatases. The mitotic phosphorylation had no apparent effect on the activity of PTP1B as measured in in vitro phosphatase assays using 32P-labeled Raytide as substrate. p34cdc2 appears not to be the mitotic PTP1B kinase, as mapping experiments showed that this enzyme phosphorylated PTP1B on a site different from that on which it was phosphorylated in vivo. These observations suggest that PTP1B may be differentially regulated through the cell cycle.