Sascha Gross

Functional interaction between nuclear inhibitor of protein phosphatase type 1 (NIPP1) and protein phosphatase type 1 (PP1) in Drosophila: consequences of over-expression of NIPP1 in flies and suppression by co-expression of PP1

The catalytic subunit of type 1 Ser/Thr protein phosphatases (PP1c) forms complexes with many proteins that target it to particular subcellular locations and regulate its activity towards specific substrates. We report the identification of a Drosophila orthologue of nuclear inhibitor of PP1 (NIPP1Dm) through interaction with PP1c in the yeast two-hybrid system. NIPP1Dm shares many properties with mammalian NIPP1 including inhibition of PP1c in vitro, binding to RNA and PP1c, and localization to nuclear speckles. However, the mechanism controlling interaction of PP1c with NIPP1 is not conserved in Drosophila. NIPP1 can function independently of PP1c as a splicing factor, but the relative importance of this function is unknown. Over-expression of NIPP1Dm in Drosophila is cell-lethal in a range of tissues and developmental stages. The effects of ectopic NIPP1Dm are suppressed by co-expression of PP1c, indicating that the only effect of ectopic NIPP1Dm is to affect PP1c function. Co-expression of NIPP1Dm and PP1c does not have any detectable physiological effect in vivo, suggesting that the NIPP1Dm-PP1c holoenzyme is not normally limiting in Drosophila. These data show that NIPP1Dm and PP1c interact in vivo and suggest that NIPP1s role as a phosphatase regulator is conserved in Drosophila.

Drosophila Uri, a PP1α binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity

BackgroundProtein phosphatase 1 (PP1) is involved in diverse cellular processes, and is targeted to substrates via interaction with many different protein binding partners. PP1 catalytic subunits (PP1c) fall into PP1α and PP1β subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1α and PP1β.ResultsURI (unconventional prefoldin RPB5 interactor) is a conserved molecular chaperone implicated in a variety of cellular processes, including the transcriptional response to nutrient signalling and maintenance of DNA integrity. We show that Drosophila Uri binds PP1α with much higher affinity than PP1β, and that this ability to discriminate between PP1c forms is conserved to humans. Most Uri is cytoplasmic, however we found some protein associated with active RNAPII on chromatin. We generated a uri loss of function allele, and show that uri is essential for viability in Drosophila. uri mutants have transcriptional defects, reduced cell viability and differentiation in the germline, and accumulate DNA damage in their nuclei.ConclusionUri is the first PP1α specific binding protein to be described in Drosophila. Uri protein plays a role in transcriptional regulation. Activity of uri is required to maintain DNA integrity and cell survival in normal development.

Essential, Overlapping and Redundant Roles of the Drosophila Protein Phosphatase 1α and 1β Genes

Protein serine/threonine phosphatase type 1 (PP1) has been found in all eukaryotes examined to date and is involved in the regulation of many cellular functions, including glycogen metabolism, muscle contraction, and mitosis. In Drosophila, four genes code for the catalytic subunit of PP1 (PP1c), three of which belong to the PP1α subtype. PP1β9C (flapwing) encodes the fourth PP1c gene and has a specific and nonredundant function as a nonmuscle myosin phosphatase. PP1α87B is the major form and contributes ∼80% of the total PP1 activity. We describe the first mutant alleles of PP1α96A and show that PP1α96A is not an essential gene, but seems to have a function in the regulation of nonmuscle myosin. We show that overexpression of the PP1α isozymes does not rescue semilethal PP1β9C mutants, whereas overexpression of either PP1α96A or PP1β9C does rescue a lethal PP1α87B mutant combination, showing that the lethality is due to a quantitative reduction in the level of PP1c. Overexpression of PP1β9C does not rescue a PP1α87B, PP1α96A double mutant, suggesting an essential PP1α-specific function in Drosophila.

The Nonmuscle Myosin Phosphatase PP1β (flapwing) Negatively Regulates Jun N-Terminal Kinase in Wing Imaginal Discs of Drosophila

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1β (PP1β). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. Here, we show genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw1, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. We show that puc expression is upregulated in wing imaginal discs mutant for flw1 and pucA251 and that this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw1 enhanced by puc. Together, we show that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs.

The non-muscle myosin phosphatase PP1beta (flapwing) negatively regulates JNK in wing imaginal discs of Drosophila

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1β (PP1β). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. Here, we show genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw1, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. We show that puc expression is upregulated in wing imaginal discs mutant for flw1 and pucA251 and that this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw1 enhanced by puc. Together, we show that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs.