Differential subcellular localization of protein phosphatase-1 alpha, gamma1, and delta isoforms during both interphase and mitosis in mammalian cells.

Protein phosphatase-1 (PP-1) is involved in the regulation of numerous metabolic processes in mammalian cells. The major isoforms of PP-1, alpha, gamma1, and delta, have nearly identical catalytic domains, but they vary in sequence at their extreme NH2 and COOH termini. With specific antibodies raised against the unique COOH-terminal sequence of each isoform, we find that the three PP-1 isoforms are each expressed in all mammalian cells tested, but that they localize within these cells in a strikingly distinct and characteristic manner. Each isoform is present both within the cytoplasm and in the nucleus during interphase. Within the nucleus, PP-1 alpha associates with the nuclear matrix, PP-1 gamma1 concentrates in nucleoli in association with RNA, and PP-1 delta localizes to nonnucleolar whole chromatin. During mitosis, PP-1 alpha is localized to the centrosome, PP-1 gamma1 is associated with microtubules of the mitotic spindle, and PP-1 delta strongly associates with chromosomes. We conclude that PP-1 isoforms are targeted to strikingly distinct and independent sites in the cell, permitting unique and independent roles for each of the isoforms in regulating discrete cellular processes.

Differential localization of myosin and myosin phosphatase subunits in smooth muscle cells and migrating fibroblasts.

Myosin II light chains (MLC20) are phosphorylated by a Ca2+/calmodulin-activated kinase and dephosphorylated by a phosphatase that has been purified as a trimer containing the delta isoform of type 1 catalytic subunit (PP1C delta), a myosin-binding 130-kDa subunit (M130) and a 20-kDa subunit. The distribution of M130 and PP1C as well as myosin II was examined in smooth muscle cells and fibroblasts by immunofluorescence microscopy and immunoblotting after differential extraction. Myosin and M130 colocalized with actin stress fibers in permeabilized cells. However, in nonpermeabilized cells the staining for myosin and M130 was different, with myosin mostly at the periphery of the cell and the M130 appearing diffusely throughout the cytoplasm. Accordingly, most M130 was recovered in a soluble fraction during permeabilization of cells, but the conditions used affected the solubility of both M130 and myosin. The PP1C alpha isoform colocalized with M130 and also was in the nucleus, whereas the PP1C delta isoform was localized prominently in the nucleus and in focal adhesions. In migrating cells, M130 concentrated in the tailing edge and was depleted from the leading half of the cell, where double staining showed myosin II was present. Because the tailing edge of migrating cells is known to contain phosphorylated myosin, inhibition of myosin LC20 phosphatase, probably by phosphorylation of the M130 subunit, may be required for cell migration.

Site-specific and temporally-regulated retinoblastoma protein dephosphorylation by protein phosphatase type 1.

pRb is dephosphorylated at mitotic exit by the type 1 serine/threonine protein phosphatases (PP1). Here we demonstrate for the first time that mitotic pRb dephosphorylation is a sequential, temporally-regulated event. We also provide evidence that the three mammalian isoforms of PP1, alpha, gamma-1, and delta, differ in their respective preferences for site-specific pRb dephosphorylation and that the mitotic and G(1) PP1-isoform counterparts exhibit differential activities towards mitotic pRb. Finally, the physiological relevance of the striking contrast between the patterns of Thr821 and Thr826 dephosphorylation, sites known to be important for disrupting binding of LXCXE-containing proteins to pRb, is addressed.

Protein phosphatase 1 isoforms in differentiating C2C12 myocytes.

In muscle protein phosphatase 1 (PP1) is involved in growth factor signal transduction and metabolic regulations. Three isoforms of the catalytic subunit are found in mammalian cells (PP1alpha, PP1gamma1 and PP1delta), with potentially different functions. We investigated the changes in the PP1 isoforms in differentiating C2C12 myoblasts. Few hours after differentiation induction the soluble PP1 activity was reversibly increased, displaying a peak at 6h. This was due to activation mainly of PP1alpha, with no change in the immunodetected protein. A further indication of PP1alpha involvement came from the observation that electroporation of inactive PP1alpha into myoblasts induced a differentiation delay of at least 24h. Subsequently, starting from 9-12 h, the activities and protein levels of all the three soluble PP1 isoforms decreased, reaching a minimum around 48 h. By this time the cells had undergone morphological changes and myosin became immunodetectable. We conclude that PP1 may be involved in myoblast differentiation, based on: 1) its higher activity in myoblasts than in myocytes, 2) the reversible activation of soluble PP1alpha during the first 6h from differentiation induction, 3) the delay in differentiation onset following electroporation of inactive PP1alpha into myoblasts.

Activation of protein phosphatase-1 isoforms and glycogen synthase kinase-3 beta in muscle from mdx mice.

Three Protein Phosphatase-1 (PP1) isoforms (PP1 alpha, PP1 gamma-1 and PP1 delta) are found in skeletal muscle. These are bound to regulatory subunits, such as inhibitor 2 (I2) in the cytosol and G in the glycogen and microsomal fractions. In vitro, the PP1-12 complex is activated by Glycogen Synthase Kinase-3 (GSK-3 or FA). We investigated the activities and protein levels of the three PP1 isoforms and of GSK-3 in muscle of mdx dystrophic mice. PP1 was assayed as phosphorylase phosphatase, in the presence of 5 nM okadaic acid (which inhibits PP2A). Peptide antibodies were produced and used to investigate PP1 alpha, PP1 gamma-1 and PP1 delta. GSK-3 was assayed using a previously described peptide. This was synthesized in a pre-phosphorylated from, which avoids the additional use of Casein Kinase II. Higher PP1 activity was assayed in the cytosol from mdx rather than from control muscles. Immunoprecipitation indicated that only PP1 alpha and PP1 gamma-1 were more active. This was most likely due to enzyme activation, since the immunodetected proteins were unchanged. On the other hand, the immunodetected PP1 delta was lower in the glycogen and microsomal fractions from mdx muscle. GSK-3 was more active in the mdx extract Selective immunoprecipitation of GSK-3 alpha and GSK-3 beta indicated that both isoforms were activated. In the case of GSK-3 beta, the immunodetected protein was also increased. The changes described herein may be related to the pathological events occurring in the mdx muscle. These include increased protein degradation and turnover, and fibre regeneration. In fact, the decreased PP1 delta may be due to protein degradation and the increased GSK-3 may be the consequence of increased protein turnover or regeneration. The apparent correlation between the increased PP1 alpha and PP1 gamma-1 activities and the increased GSK-3 may agree with the hypothesis that GSK-3 activates the newly synthesized PP1.

Activation of type-1 protein phosphatase by cdc2 kinase.

Purified cdc2 or cdc2 obtained from HeLa cells in association with p13suc1 activate inactive type-1 protein phosphatase (PP1) (catalytic subunit.inhibitor-2 complex, purified from skeletal muscle). Likewise in the case of PP1 activation by FA/GSK3, activation by cdc2 is accompanied by phosphorylation of inhibitor-2 (I2) and free I2 can be phosphorylated as well. Correlation between PP1 activation and I2 phosphorylation is suggested by the fact that both activation and phosphorylation (a) increase in parallel during incubation with cdc2, (b) decrease in parallel upon subsequent cdc2 inhibition by EDTA, and (c) are inhibited by the cdc2 inhibitor 5,6-dichlorobenzimidazole riboside. cdc2 also phosphorylates the catalytic subunit of PP1, whether in the complex with I2 or as free molecule. The activation of PP1 by cdc2 and by FA/GSK3 is compared.

Stimulation of FA and phosphatase-1 activities by insulin in 3T3-L1 cells.

The phosphatase-1 activator FA and phosphatase-1 were assayed in 3T3-L1 cells exposed to insulin. The cytosolic FA activity was transiently stimulated (7-8-fold) 1 and 2 min after exposure to 10(-8) M insulin and returned to control values within 5-10 min. Cytosolic phosphatase-1 (assayed after trypsin treatment) was activated (120-140% of controls) between 2 and 5 min and returned to control values within 10 min. Insulin effects were dose-dependent, with maximum stimulation of both activities at 10(-8) M insulin. The possibility that FA and other kinases mediate phosphatase activation by insulin is discussed.

Phosphorylation of the catalytic subunit of type-1 protein phosphatase by the v-abl tyrosine kinase.

The catalytic subunit of type-1 protein phosphatase (PP1) was phosphorylated by the tyrosine kinase v-abl as follows: (i) cytosolic PP1 was phosphorylated more (0.73 mol/mol) than PP1 obtained from the glycogen particles (0.076 mol/mol), while free catalytic subunit isolated in the active or inactive form from cytosolic PP1 was phosphorylated even less and catalytic subunit complexed with inhibitor-2 was not phosphorylated; (ii) phosphorylation stoichiometry was dependent on the concentration of PP1 and 3 h incubation at 30 degrees C was required for maximal phosphorylation; (iii) phosphorylation was on a tyrosine residue located in the C-terminal region of PP1 which is lost during proteolysis; (iv) phosphorylation did not affect enzyme activity but allowed conversion from the active to the inactive form upon incubation with inhibitor-2 of a PP1 form that in its dephospho-form did not convert.

Identification of a 68 kDa protein which copurifies with type-1 protein phosphatase as albumin.

Proteins of 60-70 kDa copurify with some preparations of type-1 or type-2 phosphatases. In our system chromatography on polylysine-Affi-Gel 10 separates a 68 kDa protein from rabbit muscle glycogen particle phosphorylase phosphatase. The separation affects neither the activity nor the size of the phosphatase. The 68 kDa protein, although pure by SDS gel electrophoresis criteria, still displays phosphatase activity of approx. 6-8 U/mg. However, rechromatography either on Bio-Gel A-0.5 m or on Blue Sepharose CL-6B followed by gel filtration shows that the activity is due to a contamination with phosphatases of type 1 and type 2, displaying a molecular mass of 35 kDa, which can be totally removed from the 68 kDa protein. The amino acid composition of the 68 kDa protein is identical to that of rabbit serum albumin, within the limits of variation for the method. Furthermore, the sequence of the 38 N-terminal amino acids is the same in the isolated 68 kDa protein and in rabbit serum albumin.

Effects of streptozotocin-diabetes, fasting and adrenaline on phosphorylase phosphatase activities of rat skeletal muscle.

The distribution of the spontaneous and trypsin-stimulated phosphorylase phosphatase activities between glycogen particles and cytosol was examined in muscle extracts obtained from rats that had been fasted, made diabetic with streptozotocin or injected with adrenaline. In all conditions the particle-bound phosphatase activities decreased, glycogen was degraded and phosphorylase was released from the particles into the cytosol. However, in fasting and diabetes (but not after adrenaline) the combined glycogen particle + cytosolic phosphatase activities decreased, indicating that the activity lost from the particles was not simply shifted to the cytosol. Fasting and diabetes (but not adrenaline) also decreased the phosphatase-activating ability of the muscle extracts, which was, at least in part, attributable to the protein kinase FA. These data indicate the presence of at least two different mechanisms affecting the phosphatase system, one modified by fasting and diabetes, the other by adrenaline.

Phosphorylase phosphatase and phosphatase activating-kinase FA in growing rat muscles.

The activities of phosphorylase phosphatase and of FA, the kinase that activates phosphatase, were measured in rat skeletal muscle from birth to 200 g body weight. Throughout this period part of phosphatase was always spontaneously active. The activity could be further increased by trypsin, but did not additionally increase when Mn2+ was present. During the first 15-20 days of life most of the phosphatase was cytosolic. Then it decreased in this fraction and more phosphatase was found in glycogen particles, to reach the adult level at about 50 g body weight. Also the activity of the kinase FA was lower for the first 10 days, then it increased attaining the adult level again at about 50 g. These results are compared to those on phosphorylase activity and glycogen level in muscle and on serum insulin during growth.

Activation of the cdc25C phosphatase in mitotic HeLa cells.

The cdc2-activator cdc25C was immunoprecipitated from HeLa cell extracts and assayed as tyrosine phosphatase (PTP) using tyrosine-phosphorylated myelin basic protein. The PTP activity was 12-fold higher in immunocomplexes from mitotic (nocodazole-arrested) than from asynchronous cells. This difference is due to enzyme activation, since the same amount of cdc25C was immunodetected in both conditions. However, mitotic cdc25C had M(r) 59,000, while a 56,000-59,000 doublet was detected in immunocomplexes from asynchronous cells. The PTP activity of mitotic cdc25C was decreased by treatment with Phosphatase-2A catalytic subunit (but not with Phosphatase-1), with re-appearance of the 56,000 polypeptide. cdc25C was also found associated with cdc2-p13-Sepharose complex and its PTP activity was 7-fold higher in samples from mitotic than from asynchronous cells. cdc25C and cdc2 co-migrated during gel filtration and the higher activity of mitotic cdc25C was retained through gel filtration.

Purification and inactivation-reactivation of phosphorylase phosphatase from the protein-glycogen complex.

Phosphorylase phosphatase purified from the protein-glycogen complex of rabbit muscle has a Mr of 34,000 by gel filtration and migrates as a single band of Mr 38,000 on sodium dodecyl sulfate gel electrophoresis, i.e., of the same size as the catalytic subunit of the sarcoplasmic complex of type 1 phosphatase (L. M. Ballou, D. L. Brautigan, and E. H. Fischer (1983) Biochemistry 22, 3393-3399). The enzyme, called PG-Ea, has a specific activity of 12,000 units/mg of protein and is essentially fully active, displaying at most a 20% further increase in activity on treatment with trypsin. As in the case of the catalytic subunit of the sarcoplasmic enzyme, tryptic attack decreases the size of PG-Ea to 33,000. PG-Ea is completely inhibited by the modulator protein (inhibitor 2) after formation of a complex of Mr 70,000. On incubation of this complex at 30 degrees C, the catalytic subunit is converted (t 1/2 = 30 min) to an inactive form (Ei) that can be reactivated by the protein kinase FA (J. R. Vandenheede, S.-D. Yang, J. Goris, and W. Merlevede (1980) J. Biol. Chem. 255, 11,768-11,774) or to a lower extent by trypsin-Mn2+. Also the trypsinized PG-Ea is inhibited by inhibitor 2, it forms with this a Mr 70,000 complex and undergoes an even faster (t 1/2 = 10 min) conversion to an Ei form that can be reactivated by the kinase FA or to a lower extent by trypsin-Mn2+. Enzymological comparison of PG-Ea and trypsinized PG-Ea with the FA-activated, isolated catalytic subunit of the sarcoplasmic phosphatase (called EaFA, E. Villa-Moruzzi, L. M. Ballou, and E. H. Fischer (1984) J. Biol. Chem. 259, 5857-5863) and with its trypsinized form shows several similarities. The most relevant of these is the very specific interaction with inhibitor 2, that takes to inactivation and allows the following reactivation by FA or by trypsin-Mn2+. However, the inactivation-reactivation patterns show also some differences, namely (i) the t 1/2 of the conversion to Ei is longer for PG-Ea than for EaFA, and (ii) the reactivation of PG-Ea and trypsinized PG-Ea with trypsin-Mn2+ is only partial. Altogether the similarity but not identity of PG-Ea and EaFA would suggest that they are either two different conformations of the same molecule or two isozymes.

Protein phosphatase-1 activation and association with the retinoblastoma protein in colcemid-induced apoptosis.

Protein phosphatase-1 (PP1) is cell cycle regulated and potentially related to apoptosis. We studied PP1 in HeLa cells exposed to colcemid, which leads first to mitotic block, then to cell death within 72 h. The soluble PP1 activity, which was low at 14 h (mitosis), was then reversibly activated (maximally around 48 h), with parallel changes in the protein levels of the alpha, gamma1 and delta PP1 isoforms. PP1 activation suggested its involvement in dephosphorylating proteins relevant to apoptosis. Among these, we examined the retinoblastoma protein (pRb). This was found hyperphosphorylated at 14 h. Hypophosphorylated pRb appeared at 24 h, increased at 48 h, and was the only form left at 72 h. PP1 was found to associate with immunoprecipitated pRb, as indicated by PP1 activity assays on the pRb-immunocomplexes. The pRb-associated PP1 activity was low at 14 h, maximal at 24 h, low again by 72 h and was due to PP1delta. The presence of active PP1 suggests its involvement in pRb dephosphorylation.

Phosphorylation of phosphatase-1alpha in cells expressing v-src.

Phosphatase-1 (PP1) is phosphorylated "in vitro" by the tyrosine-kinases c-src, v-src and v-abl. In the case of src, this induces enzyme inactivation. We investigated whether in NIH-3T3 cells expressing v-src (A4 cells) PP1 was phosphorylated on Tyr and inactivated. In mammalian cells, three PP1 isoforms are present: PP1alpha, PP1gamma1 and PP1delta. In A4 cells the three PP1 isoforms were all phosphorylated on Ser, but only PP1alpha was also phosphorylated on Tyr. A lower level of PP1 phosphorylation, and on Ser only, was found also in wild-type NIH-3T3 cells. In A4 cells most of Tyr-phosphorylated PP1alpha was cytosolic. Also the PP1 activity was decreased in the cytosol of the A4 cells. Assay of the three immunoprecipitated PP1 isoforms indicated that only PP1alpha was inactivated. Altogether the data suggest that PP1alpha might be a target of v-src "in vivo".

Glycogen-bound type-1 phosphatase: isolation and dissociation of a complex containing undegraded G-subunit.

A high molecular mass type-1 phosphatase complex can be isolated from muscle glycogen particles by a fast procedure that preserves the glycogen-binding subunit of phosphatase called G from proteolysis. G can be dissociated from such complex by ion exchange chromatography on FPLC SI column, with recovery of unproteolyzed G completely separated from phosphatase catalytic subunit.

Phosphorylation of the inhibitor-2 of protein phosphatase-1 by cdc2-cyclin B and GSK3.

cdc2-cyclin B activates protein phosphatase-1 (PP1) "in vitro", phosphorylates both catalytic subunit and inhibitor-2 (I2) and both processes are inhibited by a cdc2-inhibitory peptide. We compared the phosphorylation of I2 by cdc2-cyclin B and by the PP1-activator Glycogen Synthase Kinase 3 (GSK3). Each kinase introduced less than 0.1 mol phosphate/mol into I2 bound to PP1 and the same two tryptic phosphopeptides were obtained from I2, which contained phospho-T only. The same results were obtained also with isolated I2 phosphorylated by GSK3. Since GSK3 phosphorylates only T-72, cdc2-cyclin B is also likely to phosphorylate this site. This was confirmed by using I2 that had been mutated at this site. On the other hand cdc2-cyclin B introduced up to 0.8 mol/mol phosphate into isolated I2 and four phosphopeptides were obtained. The two new peptides contained phospho-T and one of them also phospho-S. These data indicate the presence of at least one T and one S that are phosphorylated only by cdc2-cyclin B and are accessible on isolated I2 only.