Interactions Between a Minimal Protein Serine/Threonine Phosphatase and Its Phosphopeptide Substrate Sequence

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The cDNA for casein kinase 1 (CK1) of Plasmodium falciparum was cloned, sequenced, and expressed in bacteria. The single major open reading frame of the 1.2-kilobase pair cDNA coded for a 324-amino acid polypeptide of ϳ37 kDa, the predicted sequence of which showed strong identity with known CK1 isoforms. The purified recombinant enzyme exhibited properties characteristic of CK1, such as inhibition by CK1-7, the ability to phosphorylate a highly specific peptide substrate, and a strong preference for ATP over GTP. A casein kinase activity, partially purified from soluble extracts of P. falciparum by affinity chromatography through CK1-7 columns displayed identical properties. The activity showed a stage-specific expression in the parasite, in the order trophozoite > ring > > schizont. Northern analysis indicated the existence of two major CK1 mRNAs, 2.4 and 3.2 kilobase pairs long, the levels of which were in the order ring > schizont > trophozoite. Mutagenesis of recombinant CK1 defined important amino acid residues and their potential role in the conformation of the enzyme. The malarial CK1 appeared to be the one of the smallest and perhaps the most primitive CK1 enzymes known, containing little sequence information beyond the minimal catalytic domain.The parasitic protozoan, Plasmodium falciparum, is the causative agent of malaria throughout the world and is responsible for an annual death toll of nearly 3 million, the majority of which are children and pregnant mothers (1). However, tools available to control malaria are inadequate, and drug-resistant strains are widespread; moreover, the immediate prospect of a useful vaccine is uncertain. Thus, there is an urgent need to obtain fundamental knowledge about the various cellular processes of P. falciparum at the molecular level, so that susceptible targets can be identified. With this long-term goal in mind, we have initiated studies of the signal transduction system in P. falciparum. Since reversible protein phosphorylation and dephosphorylation constitute a major mechanism of signal transduction (2), one of our immediate goals has been to characterize the various protein kinases in P. falciparum. In this paper, we report the characterization, expression, stagespecific regulation, and mutational analysis of P. falciparum casein kinase 1 (PfCK1). 1 Casein kinase-1 and -2 (CK1 and CK2) are multipotential Ser/Thr protein kinases, originally purified from rabbit reticulocyte lysates using casein as substrate (reviewed in Refs. 3 and 4). In the subsequent years, both enzymes were shown to phosphorylate, and thus regulate, a wide variety of cellular proteins. The sequence alignment of CK1 genes of various organisms and deletion analysis of recombinant yeast CK1 have recently resulted in the delineation of the following domains in the prototype 45-kDa yeast enzyme (summarized in Refs. 5 and 6): an N-terminal catalytic domain of about 300 amino acids followed by a 12-residue stretch conserved among some forms but not in others; a hydrophilic 85-residue segment predi...

Section: Discussionmentioning

confidence: 99%

“…Section 1734 solely to indicate this fact. A preliminary account of this work was presented at the Molecular Parasitology Meeting VII in Woods Hole, MA (September [15][16][17][18][19]1996).…”

Section: Methodsmentioning

confidence: 99%

Identification, Cloning, and Mutational Analysis of the Casein Kinase 1 cDNA of the Malaria Parasite, Plasmodium falciparum

Barik

Taylor

Chakrabarti

1997

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“…The PP1␣ catalytic core (residues 41-269) was expressed in bacteria and purified as described previously (15). Recombinant human inhibitor-1 was expressed, purified, and phosphorylated as described by Connor et al (16).…”

Section: Methodsmentioning

confidence: 99%

Importance of the β12-β13 Loop in Protein Phosphatase-1 Catalytic Subunit for Inhibition by Toxins and Mammalian Protein Inhibitors

Connor

Kleeman

Barik

et al. 1999

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Type-1 protein serine/threonine phosphatases (PP1) are uniquely inhibited by the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and nuclear inhibitor of PP1 (NIPP-1). In addition, several natural compounds inhibit both PP1 and the type-2 phosphatase, PP2A. Deletion of C-terminal sequences that included the ␤12-␤13 loop attenuated the inhibition of the resulting PP1␣ catalytic core by I-1, I-2, NIPP-1, and several toxins, including tautomycin, microcystin-LR, calyculin A, and okadaic acid. Substitution of C-terminal sequences from the PP2A catalytic subunit produced a chimeric enzyme, CRHM2, that was inhibited by toxins with doseresponse characteristics of PP1 and not PP2A. However, CRHM2 was insensitive to the PP1-specific inhibitors, I-1, I-2, and NIPP-1. The anticancer compound, fostriecin, differed from other phosphatase inhibitors in that it inhibited wild-type PP1␣, the PP1␣ catalytic core, and CRHM2 with identical IC 50 . Binding of wild-type and mutant phosphatases to immobilized microcystin-LR, NIPP-1, and I-2 established that the ␤12-␤13 loop was essential for the association of PP1 with toxins and the protein inhibitors. These studies point to the importance of the ␤12-␤13 loop structure and conformation for the control of PP1 functions by toxins and endogenous proteins.Type-1 protein serine/threonine phosphatases (PP1) 1 are expressed in all eukaryotic cells and have been implicated in the control of a variety of physiological processes, including carbohydrate and lipid metabolism, protein synthesis, and gene transcription (1, 2). PP2A, the major type-2 protein serine/ threonine phosphatase, shares nearly 50% sequence identity with the PP1 catalytic subunit with most of this being in sequences that organize the three-dimensional structure of the catalytic site (3). Microcystin-LR and other toxins that inhibit PP1 activity also inhibit PP2A, emphasizing the shared structural determinants at or near the catalytic site that mediate phosphatase inhibition by these natural compounds (4).Despite these similarities between the two phosphatases, cellular mechanisms that regulate PP1 and PP2A show a high degree of specificity. For instance, the mammalian proteins, inhibitor-1 (I-1), inhibitor-2 (I-2), and the nuclear inhibitor NIPP-1 uniquely inhibit PP1 activity. Moreover, these PP1 inhibitors are regulated by reversible phosphorylation so that they can modulate PP1 activity in response to hormonal stimuli. Physiological studies suggest that PP1 inhibitors function as molecular switches to control cellular signaling pathways (5). For example, I-1 and its structural homologue, DARPP-32 (dopamine-and cAMP-regulated phosphoprotein of apparent M r 32,000), once phosphorylated by cAMP-dependent protein kinase, inhibit PP1 activity to elevate and maintain cellular proteins in their phosphorylated state. In this manner, I-1 and DARPP-32 amplify and prolong cAMP signals. The importance of PP1 inhibitors in cAMP signaling was highlighted by the disruption of the mouse DARPP-32 gene (6), which severely atten...

“…Prior studies (30,31) suggested that extensive deletions of C-terminal sequences impaired or destabilized PP1␣ activity. Although recombinant PP1␣-(1-297) demonstrated phosphorylase phosphatase activity equivalent to that of full-length PP1␣, PP1␣-(1-276) was not expressed in bacteria.…”

Section: Purification Of Recombinant Pp1 Catalytic Subunits-mentioning

confidence: 99%

Importance of a Surface Hydrophobic Pocket on Protein Phosphatase-1 Catalytic Subunit in Recognizing Cellular Regulators

Gibbons¹,

Weiser

Shenolikar

2005

Cellular functions of protein phosphatase-1 (PP1), a major eukaryotic serine/threonine phosphatase, are defined by the association of PP1 catalytic subunits with endogenous protein inhibitors and regulatory subunits. Many PP1 regulators share a consensus RVXF motif, which docks within a hydrophobic pocket on the surface of the PP1 catalytic subunit. Although these regulatory proteins also possess additional PP1-binding sites, mutations of the RVXF sequence established a key role of this PP1-binding sequence in the function of PP1 regulators. WT PP1␣, the C-terminal truncated PP1␣-(1-306), a chimeric PP1␣ containing C-terminal sequences from PP2A, another phosphatase, PP1␣-(1-306) with the RVXF-binding pocket substitutions L289R, M290K, and C291R, and PP2A were analyzed for their regulation by several mammalian proteins. These studies established that modifications of the RVXF-binding pocket had modest effects on the catalytic activity of PP1, as judged by recognition of substrates and sensitivity to toxins. However, the selected modifications impaired the sensitivity of PP1 to the inhibitor proteins, inhibitor-1 and inhibitor-2. In addition, they impaired the ability of PP1 to bind neurabin-I, the neuronal regulatory subunit, and G M , the skeletal muscle glycogen-targeting subunit. These data suggested that differences in RVXF interactions with the hydrophobic pocket dictate the affinity of PP1 for cellular regulators. Substitution of a distinct RVXF sequence in inhibitor-1 that enhanced its binding and potency as a PP1 inhibitor emphasized the importance of the RVXF sequence in defining the function of this and other PP1 regulators. Our studies suggest that the diversity of RVXF sequences provides for dynamic physiological regulation of PP1 functions in eukaryotic cells.Early studies by Ingebritsen and Cohen (1, 2) classified mammalian protein serine/threonine phosphatases into two major groups, type-1 and type-2, based in large part on their sensitivity to two endogenous protein inhibitors, inhibitor-1 (I-1) 1 and inhibitor-2 (I-2). Type-I protein serine/threonine phosphatase or protein phosphatase-1 (PP1), displays unique and potent inhibition by nanomolar concentrations of I-1 and I-2. In contrast, PP2A and other type-2 protein phosphatases are essentially resistant to these inhibitor proteins. To date, more than 50 PP1-interacting proteins have been identified (3). Some, like I-1 and I-2, function as PP1 inhibitors, whereas others represent targeting subunits that direct subcellular localization and substrate recognition by multiprotein complexes containing PP1 catalytic subunits. Remarkably, PP1 and PP2A catalytic subunits share nearly 50% primary sequence identity, which may account for their overlapping substrate specificity in vitro (1). However, all PP1 regulators thus far analyzed demonstrate the unique ability displayed by I-1 and I-2 to selectively associate with PP1 and modify its catalytic function.Detailed structure-function analyses of I-1 (4) and its neuronal homologue, DARPP-32 (5), first hig...