Protein phosphorylation influences proteolytic cleavage and kinase substrate properties exemplified by analysis of in vitro phosphorylated Plasmodium falciparum glideosome-associated protein 45 by nano-ultra performance liquid chromatography-tandem mass spectrometry.
Plasmodium falciparum glideosome-associated protein 45 (PfGAP45) was in vitro phosphorylated by P. falciparum calcium-dependent protein kinase (PfCDPK1) and digested using the four proteases trypsin, chymotrypsin, AspN, and elastase. Subsequently, phosphopeptide enrichment using Ga(III) immobilized metal affinity chromatography (IMAC) was performed. The resulting fractions were analyzed using ultra performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS), resulting in the identification of a total of nine phosphorylation sites: Ser31, Ser89, Ser103, Ser109, Ser121, Ser149, Ser156, Thr158, and Ser173. During in-depth analyses of the detected phosphopeptides, it was observed that phosphorylation alters the properties of PfGAP45 as kinase and protease substrate. The closely adjacent phosphorylation sites Ser156 (major site) and Thr158 (minor site) were analyzed in detail because at first glance the specific proteases gave highly variable results with respect to the relative abundance of these sites. It was observed that (i) formation of pSer156 and pThr158 was mutually exclusive and (ii) phosphorylation at Ser156 or Thr158 interfered specifically with proteolysis by chymotrypsin or trypsin, respectively. The latter effect was studied in detail using synthetic phosphopeptides carrying either pSer156 or pThr158 as substrate for chymotrypsin or trypsin, respectively.
Winter
Dominic
D
Molecular Structure Analysis, German Cancer Research Center, D-69120 Heidelberg, Germany.
Kugelstadt
Dominik
D
Seidler
Joerg
J
Kappes
Barbara
B
Lehmann
Wolf D
WD
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis / Q000378:metabolism; D010769:Phosphothreonine / Q000032:analysis / Q000378:metabolism; D010963:Plasmodium falciparum / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D008565:Membrane Proteins; D015800:Protozoan Proteins; C509918:glideosome-associated protein 45, Plasmodium falciparum; D010769:Phosphothreonine; D010768:Phosphoserine; D011494:Protein Kinases; C531238:calcium-dependent protein kinase-1, Plasmodium falciparum; D010447:Peptide Hydrolases