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M Bollen

Publications and source records attributed to M Bollen.

At least 37 records · Page 2Linked to original sources
PubMed1997

Identification of sds22 as an inhibitory subunit of protein phosphatase-1 in rat liver nuclei.

sds22 was originally identified in yeast as a regulator of protein phosphatase-1 that is essential for the completion of mitosis. We show here that a structurally related mammalian polypeptide (41.6 kDa) is part of a 260-kDa species of protein phosphatase-1. This holoenzyme, designated PP-1N(sds22), could be immunoprecipitated with sds22 antibodies and was retained by microcystin-Sepharose. PP-1N(sds22) is a latent phosphatase, but its activity could be revealed by the proteolytic destruction of the noncatalytic subunit(s). PP-1N(sds22) accounted for only 5-10% of the total activity of PP-1 in rat liver nuclear extracts. A synthetic 22-mer peptide, corresponding to a leucine-rich repeat of sds22, specifically inhibited the catalytic subunit of PP-1, showing that at least part of the latency stems from the interaction of the sds22 repeat(s) with PP-1C.

Animals↗
PubMed1997

Association of brain protein phosphatase 1 with cytoskeletal targeting/regulatory subunits.

Protein phosphatase 1 catalytic subunit (PP1C) is highly enriched in isolated rat postsynaptic densities. Gel overlay analyses using digoxigenin (DIG)-labeled PP1C revealed four major rat brain PP1C-binding proteins (PP1bps) with molecular masses of approximately 216, 175, 134, and 75 kDa, which were (1) more abundant in brain than other rat tissues; (2) differentially expressed in microdissected brain regions; and (3) enriched in isolated cortex postsynaptic densities. PP1bp175, PP1bp134, PP1bp75, and PP1C were partially released from forebrain particulate extracts by incubation at low ionic strength, which destabilizes the actin cytoskeleton. Size-exclusion chromatography of solubilized extracts separated two main PP1 activities (approximately 600 and approximately 100 kDa). PP1bps and PP1C gamma1 were enriched in the approximately 600-kDa peak, but PP1C beta was enriched in the approximately 100-kDa peak. Furthermore, PP1bp175 and PP1bp134 exhibited lower binding of recombinant DIG-PP1C beta than recombinant DIG-PP1C gamma1 or DIG-PP1C alpha. Solubilized PP1bp175 and PP1bp134 interact with PP1C under native conditions, because they both (1) coeluted from size-exclusion and ion-exchange columns; (2) bound to microcystin-LR-Sepharose; and (3) coprecipitated using PP1C antibodies. Trypsinolysis of the approximately 600-kDa form of PP1 increased phosphorylase a phosphatase activity approximately fourfold, suggesting that interaction of PP1C with these PP1bps modulates its activity. Thus, brain PP1 activity is likely targeted to the cytoskeleton, including postsynaptic densities, by isoform-selective binding of PP1C to these targeting/regulatory subunits, contributing to the specificity of its physiological roles.

Animals↗
PubMed1996

Threonine autophosphorylation and nucleotidylation of the hepatic membrane protein PC-1.

The membrane protein plasma-cell-differentiation antigen 1 (PC-1) has been described as a phosphodiesterase-I/nucleotide pyrophosphatase and as an autophosphorylating protein kinase. It has been suggested, however, that PC-1 is not a real protein kinase and that the autophosphorylated enzyme represents a nucleotidylated derivative, which is formed on Thr238 (murine PC-1) as a catalytic intermediate during ATP hydrolysis [Belli, S.I., Mercuri, F.A., Sali, A.& Goding, J.W. (1995) Eur. J. Biochem. 228, 669-676]. We have investigated the proposed multifunctional role of PC-1 and show here that ATP hydrolysis and autophosphorylation represent two distinct catalytic reactions. The enzyme was radiolabeled when various concentrations (1-260 microM) of [alpha-32P]ATP or [alpha-32P]ADP, but not [gamma-32P]ATP, were used as substrates for the formation of the pyrophosphatase catalytic intermediate, especially in the presence of imidazole, which interferes with the hydrolysis of the nucleotidylated enzyme. In contrast, autoradiography revealed autophosphorylation only with [gamma-32P]ATP as the phosphoryl donor, and autophosphorylation has been shown to occur only at ATP concentrations below 5 microM. Autophosphorylation could also be differentiated from nucleotidylation by its higher resistance to alkaline treatment and its more basic pH optimum. An intestinal nucleotide pyrophosphatase with a structurally related catalytic site could not be autophosphorylated, which shows that autophosphorylation is not an intrinsic property of the nucleotide pyrophosphatase reaction. Autophosphorylation of PC-1 was associated with inactivation of its phosphodiesterase-I/nucleotide-pyrophosphatase activity. We propose that autophosphorylation of PC-1 on Thr238 at low ATP concentrations serves as an autoregulatory mechanism that makes Thr238 unavailable for participation in the hydrolysis of extracellular nucleotides when they become scarce.

Adenine Nucleotides↗
PubMed1996

Characterization of a ribosomal inhibitory polypeptide of protein phosphatase-1 from rat liver.

About 4% of the spontaneous phosphorylase phosphatase activity in a rat liver extract was associated with the ribosomal fraction and stemmed from both protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A). However, after repeated washing, only PP-1 remained bound to the ribosomes. The activity of ribosome-associated PP-1 (PP-1R) was partially latent and could be increased 2-3-fold by incubation with trypsin and an additional 50% by incubation with low concentrations of exogenous type-1 catalytic subunit. In contrast, incubation of the ribosomal fraction with MgATP resulted in a 50% drop in the activity of PP-1R. We have purified from a ribosomal extract a basic polypeptide (pI > or = 10.5) of 23 kDa that potently inhibited PP-1. This ribosomal inhibitor of PP-1, termed RIPP-1, was at least 30-times less efficient in inhibiting other major Ser/Thr protein phosphatases (PP-2A, PP-2B and PP-2C). RIPP-1 was identified as a non-competitive inhibitor of PP-1 with a substrate-dependent potency. The lowest Ki (approximately 20 nM) was obtained with phosphorylase and myelin basic protein as substrates. Besides instantaneously inhibiting the type-1 catalytic subunit, RIPP-1 also converted the catalytic subunit in a time-dependent manner (t 1/2 = 45 min at 25 degrees C) into a less active conformation. Unlike the inhibition, this slow inactivation was not reversed by the removal of RIPP-1. We propose that RIPP-1 accounts, at least in part, for the latency of PP-1R.

Amino Acid Sequence↗
PubMed1996

Activation of hepatic acetyl-CoA carboxylase by glutamate and Mg2+ is mediated by protein phosphatase-2A.

The activation of hepatic acetyl-CoA carboxylase by Na(+)-cotransported amino acids such as glutamine has been attributed mainly to the stimulation of its dephosphorylation by accumulating dicarboxylic acids, e.g. glutamate. We report here on a hepatic species of protein phosphatase-2A that activates acetyl-CoA carboxylase in the presence of physiological concentrations of glutamate or Mg2+ and, under these conditions, accounts for virtually all the hepatic acetyl-CoA carboxylase phosphatase activity. Glutamate also stimulated the dephosphorylation of a synthetic pentadecapeptide encompassing the Ser-79 phosphorylation site of rat acetyl-CoA carboxylase, but did not affect the dephosphorylation of other substrates such as phosphorylase. Conversely, protamine, which stimulated the dephosphorylation of phosphorylase, inhibited the activation of acetyl-CoA carboxylase. A comparison with various species of muscle protein phosphatase-2A showed that the stimulatory effects of glutamate and Mg2+ on the acetyl-CoA carboxylase phosphatase activity are largely mediated by the regulatory A subunit. Glutamate and Mg2+ emerge from our study as novel regulators of protein phosphatase-2A when acting on acetyl-CoA carboxylase.

AMP-Activated Protein Kinases↗
PubMed1996

Time-dependent pseudo-activation of hepatic glycogen synthase b by glucose 6-phosphate without involvement of protein phosphatases.

During a 30 min incubation at 25 degrees C in the presence of 5-10 mM glucose 6-phosphate, pure glycogen-bound glycogen synthase b from dog liver was progressively converted into a form that was fully catalytically active in the presence of 10 mM Na2SO4 plus 0.5 mM glucose 6-phosphate. The latter enzyme was unlike synthase a (which does not require glucose 6-phosphate for activity), and unlike synthase b (which is strongly inhibited by sulphate). The conversion was insensitive to various inhibitors of Ser/Thr-protein phosphatases and alkaline phosphatases, and was therefore termed 'pseudo-activation'. Kinetically, pseudo-activation increased the V(max) 4-fold without affecting the K(m) for the substrate UDP-glucose. Pseudo-activation appeared to be an irreversible process, but several lines of evidence argue against a limited proteolysis. Pseudo-activation of glycogen synthase occurred also readily in a rat liver cytosol, but it was not observed with purified synthase from skeletal muscle. These observations have important implications for the assay of liver gycogen-synthase phosphatase; the possible physiological implications remain to be explored.

Animals↗
PubMed1996

Modulation of basal hepatic glycogenolysis by nitric oxide.

We perfused livers from fed rats with a balanced salt solution containing 1 mmol/L glucose. Under these conditions a low steady rate of glycogenolysis was observed (approximately 1.7 micromol glucose equivalents/g/min; 20% of the maximal glycogenolytic activity). Nitric oxide (NO) transiently stimulated hepatic glucose production. A maximal response (on average doubling basal glucose output) was observed with 34 micromol/L NO. The same concentration of nitrite (NO2-) was ineffective. Half-maximal effects were seen at 8 to 10 micromol/L NO, irrespective of the flow direction (portocaval or retrograde). This glycogenolytic response to NO corresponded to a partial activation of phosphorylase. The NO effect was not additive to maximal stimulation of glycogenolysis (7.7 +/- 0.2 micromol hexose equivalents/g/min; n = 4) by 100 micromol/L dibutyryl cyclic adenosine monophosphate (Bt2cAMP). The requirement for activation of phosphorylase was also evidenced by the ineffectiveness of NO in phosphorylase-kinase-deficient livers of gsd/gsd rats. The NO effect was blocked by co-administration of cyclooxygenase inhibitors (50 micromol/L ibuprofen, 50 micromol/L indomethacin, or 2 mmol/L aspirin), suggesting a mediatory role of prostanoids from nonparenchymal cells. This conclusion was confirmed by the fact that NO did not activate phosphorylase in isolated hepatocytes. Moreover, NO was no longer glycogenolytic in livers perfused with Ca2+-free medium, in agreement with the known mediatory role of Ca2+ in prostanoid-mediated responses. Surprisingly, in Ca2+-free medium NO inhibited the basal glucose production. This coincided with an increased elution of cyclic guanosine monophosphate (cGMP). Inhibition of glycogenolysis by NO under these conditions was blocked by 1 mmol/L theophylline, suggestive for involvement of cGMP-stimulated cAMP phosphodiesterase. However, we could not confirm that an increase in cGMP resulted in a drop in cAMP. In conclusion, NO recruits opposing mechanisms with respect to modulation of basal hepatic glycogenolysis. In the presence of Ca2+, activation of phosphorylase with stimulation of glycogenolysis dominates. Cyclooxygenase inhibitors abolish this effect. Activation by NO of the cyclooxygenase in nonparenchymal cells is a distinct possibility. In the absence of Ca2+, inhibition of basal glycogenolysis becomes observable. It remains to be established whether this results from cGMP-mediated stimulation of hydrolysis of cAMP.

Animals↗
PubMed1996

The inhibition of the insulin receptor by the receptor protein PC-1 is not specific and results from the hydrolysis of ATP.

The membrane protein plasma cell differentiation antigen 1 (PC-1) has been purified as an inhibitor of insulin receptor tyrosine kinase activity and has been implicated in the pathogenesis of NIDDM. However, we show here that PC-1 is a general protein kinase inhibitor in vitro and that this inhibition results from the hydrolysis of ATP by the intrinsic nucleotide pyrophosphatase activity of PC-1. Thus, the inhibition diminished with increasing ATP concentrations, and it was nullified when the ATP concentration was kept constant with a regenerating system or when ATP was added repetitively. When care was taken to avoid ATP depletion, PC-1 did not affect the insulin sensitivity of insulin receptor autophosphorylation. We conclude that the reported inhibition of insulin signaling by PC-1 does not result from a direct inhibition of the insulin receptor kinase activity.

Adenosine Triphosphate↗