Abstract: PP1 (protein phosphatase 1) is a ubiquitously expressed serine/threonine-specific protein phosphatase whose activity towards different substrates appears to be mediated via binding to specific proteins that play critical regulatory and targeting roles. In the present paper we report the cloning and characterization of a new protein, termed SARP (several ankyrin repeat protein), which is shown to interact with all isoforms of PP1 by a variety of techniques. A region encompassing a consensus PP1-binding motif in SARP (K354VHF357) modulates endogenous SARP-PP1 activity in mammalian cells. This SARP-PP1 interaction motif lies partially within the first ankyrin repeat in contrast with other proteins [53BP2 (p53 binding protein 2), MYPT1/M(110)/MBS (myosin binding protein of PP1) and TIMAP (transforming growth factor beta inhibited, membrane-associated protein)], where a PP1-binding motif precedes the ankyrin repeats. Alternative mRNA splicing produces several isoforms of SARP from a single human gene at locus 11q14. SARP1 and/or SARP2 (92-95 kDa) are ubiquitously expressed in all tissues with high levels in testis and sperm, where they are shown to interact with both PP1gamma1 and PP1gamma2. SARP3 (65 kDa) is most abundant in brain where SARP isoforms interact with both PP1alpha and PP1gamma1. SARP is highly abundant in the nucleus of mammalian cells, consistent with the putative nuclear localization signal at the N-terminus. The presence of a leucine zipper near the C-terminus of SARP1 and SARP2, and the binding of mammalian DNA to SARP2, suggests that SARP1 and SARP2 may be transcription factors or DNA-associated proteins that modulate gene expression.

Abstract: Altered metabolism of the Alzheimer's amyloid precursor protein (APP) appears to be a key event in the pathogenesis of Alzheimer's disease (AD), and both altered phosphorylation and oxidative stress appear to affect the production of the toxic Abeta fragment. Our results show that altered processing of APP was observed under conditions of stress induced by sodium azide in the presence of 2-deoxy-D-glucose (2DG). As previously reported, the production of the secreted fragment of APP (sAPP) was inhibited. Using APP-GFP fusion proteins, we show that 2DG causes the accumulation/delay of APP in the endoplasmic reticulum (ER)/Golgi (G). The 751 isoform accumulated preferentially in the G, whereas the 695 isoform was blocked preferentially at the ER. This effect was augmented in the presence of sodium azide. APP subcellular distribution was also affected at the plasma membrane. The involvement of protein phosphorylation in APP subcellular localization was reinforced by the effect of drugs, such as phorbol 12-myristate 13-acetate (PMA), since APP was completely depleted from the membrane in the presence of 2DG and PMA. Thus, the hypothesis that APP is processed in a phosphorylation-dependent manner and that this may be of clinical relevance appears to hold true even under stress conditions. Our results provide evidence for a role of protein phosphorylation in APP sorting under stress conditions and contribute to the understanding of the molecular basis of AD.

Abstract: Nek2 is a cell cycle-regulated serine/threonine protein kinase that is upregulated in human cancers. Functionally, it is implicated in control of centrosome separation and bipolar spindle formation in mitotic cells and chromatin condensation in meiotic cells. Two major splice variants have been described in vertebrates, Nek2A and Nek2B, that differ in their non-catalytic carboxyl-termini. Recently, a third splice variant, Nek2C, was identified that lacks an eight amino acid internal sequence within the carboxyl-terminal domain of Nek2A. This excision occurs at the same position as the Nek2A/Nek2B splice point. As predicted from their high degree of similarity, we show here that Nek2C shares many properties with Nek2A including kinase activity, dimerization, PP1 interaction, mitotic degradation, microtubule binding and centrosome localization. Unexpectedly, though, the non-centrosomal pool of protein exhibits a marked difference in distribution for the three splice variants. Nek2C is mainly nuclear, Nek2B is mainly cytoplasmic and Nek2A is evenly distributed within nuclei and cytoplasm. Mutagenesis experiments revealed a functional bipartite nuclear localization sequence (NLS) that spans the splice site leading to Nek2C having a strong NLS, Nek2A a weak NLS and Nek2B no NLS. Finally, we identified a 28 kDa protein in nuclear extracts as a potential novel substrate of Nek2. Thus, alternative splicing provides an unusual mechanism for modulating Nek2 localization enabling it to have both nuclear and cytoplasmic functions.

Abstract: Mammalian sperm were previously shown to express the PP1gamma2 isoform of protein phosphatase 1 (PP1) as well as its regulatory proteins inhibitor 2 and glycogen synthase kinase 3. Furthermore, the development of sperm motility during transit through the epididymis correlates with changes in PP1 activity. Thus, since PP1 cellular activity is determined by the partners it binds, we embarked on a study aimed at defining the specific interactomes of PP1gamma1 and PP1gamma2 (the two known alternatively spliced variants of PP1gamma). To this end, exhaustive screens were performed on a human testis cDNA library using the yeast two-hybrid method. Among the various proteins detected, the most abundant interactors with PP1gamma2 were Nek2A and R15B. Closer sequence analysis revealed novel alternatively spliced variants of Nek2A and NIPP1, which we designated Nek2A-T and NIPP1-T, respectively. They were shown to be highly expressed in rat and human testis by Northern analysis and to result from alternative splicing events by RT-PCR. Thus, both the previously known Nek2A isoform and the novel Nek2A-T and NIPP1-T variants appear to bind PP1gamma2 in vitro (blot overlays) and in vivo by coexpression in yeast. The usefulness of testis-specific alternatively spliced proteins as targets for the development of novel therapeutic strategies for male infertility and contraception is discussed. PP1gamma2, Nek2A-T, and NIPP1-T are currently being investigated as alternatively spliced targets for signal transduction therapeutics.