Drosophila Uri, a PP1alpha binding protein, is essential for viability, maintenance of DNA integrity and normal transcriptional activity.

BACKGROUND: Protein 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 PP1alpha and PP1beta subfamilies based on sequence analysis, however very few PP1c binding proteins have been demonstrated to discriminate between PP1alpha and PP1beta. RESULTS: URI (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 PP1alpha with much higher affinity than PP1beta, 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. CONCLUSION: Uri is the first PP1alpha 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 1 alpha and 1 beta 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 PP1 alpha subtype. PP1 beta 9C (flapwing) encodes the fourth PP1c gene and has a specific and nonredundant function as a nonmuscle myosin phosphatase. PP1 alpha 87B is the major form and contributes approximately 80% of the total PP1 activity. We describe the first mutant alleles of PP1 alpha 96A and show that PP1 alpha 96A is not an essential gene, but seems to have a function in the regulation of nonmuscle myosin. We show that overexpression of the PP1 alpha isozymes does not rescue semilethal PP1 beta 9C mutants, whereas overexpression of either PP1 alpha 96A or PP1 beta 9C does rescue a lethal PP1 alpha 87B mutant combination, showing that the lethality is due to a quantitative reduction in the level of PP1c. Overexpression of PP1 beta 9C does not rescue a PP1 alpha 87B, PP1 alpha 96A double mutant, suggesting an essential PP1 alpha-specific function in Drosophila.

The nonmuscle myosin phosphatase PP1beta (flapwing) negatively regulates Jun N-terminal kinase in wing imaginal discs of Drosophila.

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1beta (PP1beta). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1alpha 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 essential role of PP1beta in Drosophila is to regulate nonmuscle myosin.

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.