1. Contexto de la investigación
1.3 Planteamiento del problema
AMPK was partially purified from rat brain and liver as described in materials and methods (section 2.12.3). The brain enzyme had a lower specific activity than the liver enzyme (0.41 ± 0.09 nmol ^^P-phosphate into SAMS peptide/min/mg protein in brain; 1.67 ± 0.85 nmol ^^P-phosphate into SAMS peptide/min/mg protein in liver), but showed a similar profile of sensitivity to increasing concentrations of AMP (Figure 4.1). The Kg for AMP determined in each case using the Wilkinson Derivation plot in the presence of 0.2 mM ATP was 1.61 ± 0.41 |iM for the brain and 3.37 ± 0.57 |iM for liver. The Kg is dependent on the AMP/ATP ratio, Carling et al (1989) measured the Kg for more extensively purified rat liver AMPK at both 0.2 mM ATP (1.4 |lM ) and 2.0 mM ATP (14 flM), which is a more physiological ATP concentration.
Brain and liver AMPK were assayed in the presence of increasing concentrations of PCr up to 200 mM (Figure 4.2). Dose-dependent inhibition of AMPK from both sources was observed in the absence of AMP but this dependence was different for brain and liver. Liver AMPK was 60% inhibited by 50 mM PCr while brain AMPK was virtually unaffected. Estimated EC5 0 for PCr for the liver enzyme was 35 mM and for brain AMPK
75 mM.
Ponticos et al (1998) reported that inhibition of antibody-purified liver AMPK by PCr occurred in the presence or absence of AMP. This was also true in the present study for the liver enzyme (Figure 4.2B). However, addition of 150 |lM AMP to the brain enzyme greatly increased the sensitivity of AMPK to PCr inhibition so that the EC5 0 decreased to
32 mM PCr whereas in the absence of AMP it was 75 mM PCr (Figure 4.2A). In the presence of 150 flM AMP the sensitivity of brain AMPK to inhibition by PCr was
Figure 4.1 AMP sensitivity of AMPK from rat brain and liver
AMPK was purified to the post mono-Q stage as described in materials and methods (see section 2.12.3) from both rat brain and liver. The two preparations were assayed for AMPK activity by the incorporation of '^“P-phosphate into SAMS peptide over 5 min incubations as described in materials and methods (see section 2 .8 .2 ), over a range of
concentrations of its allosteric activator, AMP. ATP was present at 0.2 mM in each assay. Activity was calculated as percent of control (no AMP). A. Brain AMPK activity, 100 % = 0.41 ± 0.09 nmol phosphate into SAMS peptide/min/mg protein. B. Liver AMPK activity, 100 % = 1.67 ± 0.85 nmol phosphate into SAMS peptide/min/mg protein. Each value is the mean of two or three determinations.
_ .too- 1/ ■— 2 0 0- IO(H 10.11 100.0 A M PI Ui M) . t o o - IO(M 100. A M P ! (liM) 161
Figure 4.2 Effect of phosphocreatine on AMPK activity of rat brain and liver
AMPK was purified to the post mono-Q stage as described in materials and methods (see section 2.12.3) from both rat brain and liver. The two preparations were assayed for AMPK activity by the incorporation of'^“P-phosphate into SAMS peptide as described in materials and methods (see section 2 .8 .2 ) over a range of concentrations of
phosphocreatine (PCr) in the presence or absence of 150 fiM AMP. Activity was calculated as percent of control (no PCr). A. Brain AMPK activity, in the absence of AMP, average control value 0.75 ± 0.04 nmoles phosphate into SAMS/min/mg protein, and in the presence of 150 pM AMP B. Liver AMPK activity, in the absence of AMP, average control value 2.46 ± 0.89 nmoles phosphate into SAMS/min/mg protein, and in the presence of 150 pM AMP. Each value is the mean ± sem, where appropriate, of at least three determinations.
0 u "o & >
1
i
< 125- 1004 75- 50- 25 0 o . AMP • + AMP 0 50 100 150 200 § 0 "o & >1
1 0 0^ k 75- 50- 25- 0B
o- - AMP • + AMP 0 50 100 150 200 [ phosphocreatine 1 (mM) 162indistinguishable from liver AMPK (Figure 4.2).The EC5 0 for PCr inhibition of liver
AMPK measured by Ponticos et al (1998) was approximately 15 mM [PCr. The discrepancy between this value and the values reported here may be due to the greater purity of AMPK used in the studies of Ponticos et al (1998) as this had been further purified past the DEAE sepharose step by immunoprécipitation.
Figure 4.3 shows that the sensitivity of liver AMPK to activation by increasing AMP concentrations is unchanged by the presence of 100 mM PCr (Figure 4.3B). However, brain AMPK appears to show a slight increase in sensitivity to AMP in the presence of
100 mM PCr (Figure 4.3 A).
From its initial discovery, AMPK was known to be reversibly phosphorylated and activated (Carling et al, 1986). Subsequent insensitivity to OA and experiments with purified phosphatases have led to the conclusion that PP2C is the physiological PP that dephosphorylates liver AMPK (Davies et al, 1995). Figure 4.4 shows that brain AMPK is also regulated by reversible phosphorylation, in this experiment it was dephosphorylated and inactivated (80%) by incubation with PP2C.
To make some comparison of the substrate specificity of brain and liver AMPK a range of peptide substrates were assayed with each enzyme. The consensus sequence of AMPK has been found by various groups to include a hydrophobic residue at -4 or -5 from the serine to be phosphorylated (Weekes et al, 1993, Toomey et al, 1995, Dale et al, 1995), and hydrophobic at +4 or +5 (Weekes et al, 1993, Dale et al, 1995), and a basic residue a t-3 o r-4 (Sullivan er fl/, 1994, Weekes gr aZ, 1993, Dale era/, 1995, Toomey era/, 1995). The prim ary amino acid sequence surrounding serine-79 of ACC (HMRSSMSGLHVKRR) contains the hydrophobic met at -5, a basic arg at -4, and leu and val at +4 and +5, so it is a very good substrate for AMPK.
Figure 4.3 AMP sensitivity of AMPK purified from ra t brain and liver in the presence and absence of PCr
AMPK was purified to the post mono-Q stage as described in materials and methods (see section 2.12.3) from both rat brain and liver. The two preparations were assayed for AMPK activity by the incorporation of ^“P-phosphate into SAMS peptide as described in materials and methods (see section 2.8.2) over a range of concentrations of AMP in the presence or absence of 100 mM phosphocreatine (PCr). Activity was calculated as percent of control (no AMP). A. Brain AMPK activity, control value 0.41 ± 0.09 nmoles phosphate into SAMS/min/mg protein. B. Liver AMPK activity, control value 1.67 ± 0.85 nmoles phosphate into SAMS/min/mg protein. Values are from two determinations.
;
400- 300- 200- 100 0 • 100 mM P C r o no P C r 0 50 100 150 200 [AMP] (pM)P
400- 300- 200- 100 0B
• 100 mM P C r ^ no P C r 0 50 100 150 [AMP] (pM) 200 164Figure 4.4 Déphosphorylation and inactivation of brain AMPK by PP2C
AMPK was purified from rat brain as far as the DEAE step, as described in materials and methods (section 2.12.3). AMPK was then incubated at 37°C over 30 min in the presence of recombinant PP2C in the presence of MgCl2 (PP2C will be active), and the
absence of MgCl2 (PP2C will remain inactive). At time points throughout this
incubation, aliquots were removed and diluted into buffer containing 50 mM NaF (to inhibit active phosphatase) and assayed for AMPK activity by the incorporation of ^^P- phosphate into SAMS peptide as described in section 2.8.2. Results are shown as pmol ^^P-phosphate incorporated into SAMS /min from a single determination.
II
— c/5 w c/5f.i
2 1.25n 1.0 0- 0.75- 0.50- 0.25- 0.00 0 no MgCl2 MgCl2 10 20 Time (mins) 30 165Table 4.1 Specificity of brain and liver AMPK for synthetic peptide substrates.
Peptides containing a modified consensus sequence of the residues surrounding serine 79 of ACC were synthesised by N Flinn at the School of Pharmacy, University of London (Toomey et al, 1995). Phosphorylation by DEAE purified brain or liver AMPK of each synthetic peptide shown was assayed by the incorporation of ^^P-phosphate into each peptide as described in materials and methods (see section 2 .8 .2 ). peptides were
used at a final sub-saturating concentration of 40 pM in the assay and only initial rates of phosphorylation (over the first 5 min) were measured. Peptides are referred to in the text by the four letter code shown in colour. Serine-79 is underlined. Activity is expressed as a percentage of the rate of phosphorylation of the parent peptide, HMRSSMSGLHVKRR. Values are mean ± sem. for 3 observations, 5 for SAMS.
Peptide Brain Liver
HMRSSMSGLHLVKRR 1 0 0 1 0 0 AMRSSMSGLHLVKRR 295 ±31 445 ± 155 WMRSSMSGLHLVKRR 434 ±61 444 ± 147 HFRSSMSGLHLVKRR 250 ± 36 208 ± 26 HMRSAMSGLHLVKRR 77 ±13 290 ± 45 HARSSMSGLHLVKRR 2 4 ± 15 7 ± 5 HMASSMSGLHLVKRR 15 ±15 3 ± 3
The peptides prepared by Toomey et al (1995) were based on this sequence from ACC, with alterations of the first three residues, and these were used to assay brain and liver AMPK. Both brain and liver AMPK activity increased when the his at - 6 was replaced
with an uncharged ala or a hydrophobic trp group, and when the met at -5 was replaced with the bulky, hydrophobic phe (Table 4.1). However, the arg at -4 was vital to AMPK from both tissues. Its replacement with an uncharged ala virtually abolished AMPK activity, as did replacing met at -5 with ala. The data presented in Table 4.1 shows that the substrate specificity of AMPK in liver and brain is very similar. One important exception to this is that brain AMPK phosphorylates SAMS to a similar extent to SSMS, but liver AMPK phosphorylates SAMS four times better than the brain enzyme (Table 4.1). This is hard to explain, if it is due to contaminating kinases these cannot include PKA because neither brain nor liver AMPK phosphorylated HARSSMS to any great extent (see section 1.9.1), and the other peptides are phosphorylated with similar ratios by both brain and liver AMPK. The use of SAMS as a peptide substrate is not invalidated by these data because at 200 pM SAMS the specific activities of brain and liver AMPK were equivalent. At the sub-saturating concentration of 40 pM, brain AMPK has less affinity for SAMS than liver AMPK.