Limits...
Use of a decoy peptide to purify p21 activated kinase-1 in cardiac muscle and identification of ceramide-related activation.

Ke Y, Solaro RJ - Biologics (2008)

Bottom Line: Dihydro-L-threo-sphingosine (saphingol) also had some effect on Pak1 autophosphorylation.The method we developed provides a useful tool to study Pak1 activity and regulation in the heart.Moreover, our results indicate a potential role of the sphingolipids as unique signaling molecules inducing a direct activation of Pak1 that may modulate different cardiac functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL, USA.

ABSTRACT
The p(21) activated kinase-1 (Pak1) is a serine-threonine protein kinase directly activated by Cdc42 and Rac1. In cardiac myocytes, Pak1 activation leads to dephosphorylation of cTnI and C-protein through upregulation of phosphatase-2A (PP2A). Pak1 activity is directly correlated with its autophosphorylation, which occurs upon binding to the small GTPases and to some small organic molecules as well. In this report, we describe a novel method for rapid purification of endogenous Pak1 from bovine ventricle muscle. The method is simple and easy to carry out. The purified Pak1 demonstrated autophosphorylation in vitro that was enhanced by D-erythro-sphingosine-1, N-acetyl-D-erythro-sphingosine (C(2)-ceramide), and N-hexanoyl-D-erythro-sphingosine (C(6)-ceramide). Dihydro-L-threo-sphingosine (saphingol) also had some effect on Pak1 autophosphorylation. The method we developed provides a useful tool to study Pak1 activity and regulation in the heart. Moreover, our results indicate a potential role of the sphingolipids as unique signaling molecules inducing a direct activation of Pak1 that may modulate different cardiac functions.

No MeSH data available.


Purified native Pak1 From cardiac muscle. A. Purification of endogenous Pak1 from cardiac muscle. 1. Cardiac muscle extracts resolved on a SDS page. 2. The precipitation. 3. Pak1 purified from the fractionated muscle sample. 4. The molecular weight markers. cardiac muscle extract after fractionation by 25% and 50% (NH4)2SO4 B. Detection of purified Pak1 by Western blotting analysis. Pak1 was detected as a single band with an antibody from Santa Cruz (sc-881). C. Purified Pak1 detected by mass spectrometry. Peaks match the Pak1 peptides produced by theoretical trypsin digestion. The positions of amino acids of each peptide from Pak1 were denoted in quotation: LSAIFR (490–495), ELLQHQFLK (514–522), SVIEPLPVTPTR (204–215), KELIINEILVMR (309–320), ECLQALEFLHSNQVIHR (372–388).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2727905&req=5

f2-btt-2-903: Purified native Pak1 From cardiac muscle. A. Purification of endogenous Pak1 from cardiac muscle. 1. Cardiac muscle extracts resolved on a SDS page. 2. The precipitation. 3. Pak1 purified from the fractionated muscle sample. 4. The molecular weight markers. cardiac muscle extract after fractionation by 25% and 50% (NH4)2SO4 B. Detection of purified Pak1 by Western blotting analysis. Pak1 was detected as a single band with an antibody from Santa Cruz (sc-881). C. Purified Pak1 detected by mass spectrometry. Peaks match the Pak1 peptides produced by theoretical trypsin digestion. The positions of amino acids of each peptide from Pak1 were denoted in quotation: LSAIFR (490–495), ELLQHQFLK (514–522), SVIEPLPVTPTR (204–215), KELIINEILVMR (309–320), ECLQALEFLHSNQVIHR (372–388).

Mentions: Figure 1 show results of immuno-histochemical studies in rat ventricular myocytes demonstrating high and localized expression of Pak1. The bleb in the image of Figure 1A (upper panel) indicates sarcolemmal localization. Pak 1 also localized to the nuclear membrane, Z-discs and intercalated discs (Figure 1A). To isolate the cardiac Pak1, we used bovine ventricle muscle as the source as described in the Methods. About 800 mg of muscle protein extract was obtained from 200 g of the frozen ventricle muscle. Therefore, the yield of muscle extract from the frozen tissue is about 0.4%. Figure 1B illustrates the approach we used for affinity purification of the Pak1 as described in the Methods. The peptide linked affinity column was loaded with 5 mg of the protein extracted from the muscle sample. Data in Figure 2A demonstrate that Pak1 is the major component in the eluted fraction as indicated by the resolution of the samples by SDS-PAGE with Coomassie brilliant blue staining (Figure 3A, lane 3). Ammonia sulfate fractionation decreased amount of some proteins with the molecular weight larger and smaller than Pak1 (Figure 3A). Western blotting analysis (Figure 2B) using an antibody (sc-881) identified the bands shown in Figure 2A as Pak1. The affinity purification procedure yielded about 200 μg of total protein from the 5 mg muscle protein extract prepared from the frozen ventricle with a yield of 4%. Therefore, the yield of final affinity purification product from the frozen ventricle is about 0.016% (w/w). Pak1 bound to the matrix was eluted with 3 mg of HA peptide that displace the decoy peptide from the matrix. The HA peptide in the eluant was separated from Pak1 protein by use of dialysis tubing permeable to proteins smaller than 12–14 kD (Figure 1B). The bound Pak1 can also be eluted with Glycine (0.1 M, pH2.0). However, elution with glycine sometimes produces an extra protein bands with molecular weight of 16 kD. Mass spectrometry analysis indicated that the major MS peaks from the digested protein purified matches with Pak1 peptides after trypsin digestion (Figure 2C).


Use of a decoy peptide to purify p21 activated kinase-1 in cardiac muscle and identification of ceramide-related activation.

Ke Y, Solaro RJ - Biologics (2008)

Purified native Pak1 From cardiac muscle. A. Purification of endogenous Pak1 from cardiac muscle. 1. Cardiac muscle extracts resolved on a SDS page. 2. The precipitation. 3. Pak1 purified from the fractionated muscle sample. 4. The molecular weight markers. cardiac muscle extract after fractionation by 25% and 50% (NH4)2SO4 B. Detection of purified Pak1 by Western blotting analysis. Pak1 was detected as a single band with an antibody from Santa Cruz (sc-881). C. Purified Pak1 detected by mass spectrometry. Peaks match the Pak1 peptides produced by theoretical trypsin digestion. The positions of amino acids of each peptide from Pak1 were denoted in quotation: LSAIFR (490–495), ELLQHQFLK (514–522), SVIEPLPVTPTR (204–215), KELIINEILVMR (309–320), ECLQALEFLHSNQVIHR (372–388).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2727905&req=5

f2-btt-2-903: Purified native Pak1 From cardiac muscle. A. Purification of endogenous Pak1 from cardiac muscle. 1. Cardiac muscle extracts resolved on a SDS page. 2. The precipitation. 3. Pak1 purified from the fractionated muscle sample. 4. The molecular weight markers. cardiac muscle extract after fractionation by 25% and 50% (NH4)2SO4 B. Detection of purified Pak1 by Western blotting analysis. Pak1 was detected as a single band with an antibody from Santa Cruz (sc-881). C. Purified Pak1 detected by mass spectrometry. Peaks match the Pak1 peptides produced by theoretical trypsin digestion. The positions of amino acids of each peptide from Pak1 were denoted in quotation: LSAIFR (490–495), ELLQHQFLK (514–522), SVIEPLPVTPTR (204–215), KELIINEILVMR (309–320), ECLQALEFLHSNQVIHR (372–388).
Mentions: Figure 1 show results of immuno-histochemical studies in rat ventricular myocytes demonstrating high and localized expression of Pak1. The bleb in the image of Figure 1A (upper panel) indicates sarcolemmal localization. Pak 1 also localized to the nuclear membrane, Z-discs and intercalated discs (Figure 1A). To isolate the cardiac Pak1, we used bovine ventricle muscle as the source as described in the Methods. About 800 mg of muscle protein extract was obtained from 200 g of the frozen ventricle muscle. Therefore, the yield of muscle extract from the frozen tissue is about 0.4%. Figure 1B illustrates the approach we used for affinity purification of the Pak1 as described in the Methods. The peptide linked affinity column was loaded with 5 mg of the protein extracted from the muscle sample. Data in Figure 2A demonstrate that Pak1 is the major component in the eluted fraction as indicated by the resolution of the samples by SDS-PAGE with Coomassie brilliant blue staining (Figure 3A, lane 3). Ammonia sulfate fractionation decreased amount of some proteins with the molecular weight larger and smaller than Pak1 (Figure 3A). Western blotting analysis (Figure 2B) using an antibody (sc-881) identified the bands shown in Figure 2A as Pak1. The affinity purification procedure yielded about 200 μg of total protein from the 5 mg muscle protein extract prepared from the frozen ventricle with a yield of 4%. Therefore, the yield of final affinity purification product from the frozen ventricle is about 0.016% (w/w). Pak1 bound to the matrix was eluted with 3 mg of HA peptide that displace the decoy peptide from the matrix. The HA peptide in the eluant was separated from Pak1 protein by use of dialysis tubing permeable to proteins smaller than 12–14 kD (Figure 1B). The bound Pak1 can also be eluted with Glycine (0.1 M, pH2.0). However, elution with glycine sometimes produces an extra protein bands with molecular weight of 16 kD. Mass spectrometry analysis indicated that the major MS peaks from the digested protein purified matches with Pak1 peptides after trypsin digestion (Figure 2C).

Bottom Line: Dihydro-L-threo-sphingosine (saphingol) also had some effect on Pak1 autophosphorylation.The method we developed provides a useful tool to study Pak1 activity and regulation in the heart.Moreover, our results indicate a potential role of the sphingolipids as unique signaling molecules inducing a direct activation of Pak1 that may modulate different cardiac functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL, USA.

ABSTRACT
The p(21) activated kinase-1 (Pak1) is a serine-threonine protein kinase directly activated by Cdc42 and Rac1. In cardiac myocytes, Pak1 activation leads to dephosphorylation of cTnI and C-protein through upregulation of phosphatase-2A (PP2A). Pak1 activity is directly correlated with its autophosphorylation, which occurs upon binding to the small GTPases and to some small organic molecules as well. In this report, we describe a novel method for rapid purification of endogenous Pak1 from bovine ventricle muscle. The method is simple and easy to carry out. The purified Pak1 demonstrated autophosphorylation in vitro that was enhanced by D-erythro-sphingosine-1, N-acetyl-D-erythro-sphingosine (C(2)-ceramide), and N-hexanoyl-D-erythro-sphingosine (C(6)-ceramide). Dihydro-L-threo-sphingosine (saphingol) also had some effect on Pak1 autophosphorylation. The method we developed provides a useful tool to study Pak1 activity and regulation in the heart. Moreover, our results indicate a potential role of the sphingolipids as unique signaling molecules inducing a direct activation of Pak1 that may modulate different cardiac functions.

No MeSH data available.