The retinoblastoma protein {pll0 nB) interacts with many cellular proteins in complexes potentially important for its growth-suppressing [unction. We have developed and used an improved version of the yeast two-hybrid system to isolate human cDNAs encoding proteins able to bind pll0 RB. One clone encodes a novel type 1 protein phosphatase catalytic subunit (PP-la2), which differs from the originally defined PP-lc~ by an amino-terminal l 1-amino-acid insert. In vitro-binding assays demonstrated that PP-lc~ isoforms preferentially bind the hypophosphorylated form of p ll0 RB. Moreover, similar pll0 RB sequences are required for binding PP-lc~2 and SV40 large T antigen. Cell cycle synchrony experiments revealed that this association occurs from mitosis to early Gv The implications of these findings on the regulation of both proteins are discussed.

/pdf/the-retinoblastoma-protein-associates-with-the-protein-5cuo8mmht8.pdf

The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit.

Protein phosphatase 1 (PP1) is a major eukaryotic protein serine/threonine phosphatase that regulates an enormous variety of cellular functions through the interaction of its catalytic subunit (PP1c) with over fifty different established or putative regulatory subunits. Most of these target PP1c to specific subcellular locations and interact with a small hydrophobic groove on the surface of PP1c through a short conserved binding motif--the RVxF motif--which is often preceded by further basic residues. Weaker interactions may subsequently enhance binding and modulate PP1 activity/specificity in a variety of ways. Several putative targeting subunits do not possess an RVxF motif but nevertheless interact with the same region of PP1c. In addition, several 'modulator' proteins bind to PP1c but do not possess a domain targeting them to a specific location. Most are potent inhibitors of PP1c and possess at least two sites for interaction with PP1c, one of which is identical or similar to the RVxF motif. Regulation of PP1c in response to extracellular and intracellular signals occurs mostly through changes in the levels, conformation or phosphorylation status of targeting subunits. Understanding of the mode of action of PP1c complexes may facilitate development of drugs that target particular PP1c complexes and thereby modulate the phosphorylation state of a very limited subset of proteins.

Protein phosphatase 1 – targeted in many directions

On target with a new mechanism for the regulation of protein phosphorylation

A Protein Complex Network of Drosophila melanogaster

Novel protein serine/threonine phosphatases: variety is the spice of life.

Protein serine/threonine phosphatases; an expanding family

The amino acid sequence of a novel mammalian protein phosphatase, termed PPX (and designated PPP4 in the human genome nomenclature), has been deduced from the cDNA and shown to be 65% identical to PP2A alpha and PP2A beta and 45% identical to PPI isoforms, the predicted molecular mass being 35 kDa PPX was expressed in the baculovirus system Its substrate specificity and sensitivity to the inhibitors, okadaic acid and microcystin, were similar (but not identical) to the catalytic subunit of PP2A However, PPX did not bind the 65 kDa regulatory subunit of PP2A The intracellular localization of PPX was investigated by immunofluorescence using two different antibodies raised against bacterially expressed PPX and a PPX-specific peptide These showed that although PPX was distributed throughout the cytoplasm and the nucleus, intense staining occurred at centrosomes The centrosomal staining was apparent in interphase and at all stages of mitosis, except telophase In contrast, antibodies directed against bacterially expressed PP2A were not specifically localized to centrosomes The human autoantibody #5051, which stains the pericentriolar material, colocalizes with PPX antibodies, suggesting that PPX may play a role in microtubule nucleation

PPX, a novel protein serine/threonine phosphatase localized to centrosomes.

The protein serine/threonine phosphatase 4 (PP4), which localises to centrosomes/spindle pole bodies in human cells, is shown to exhibit a similar localisation in Drosophila cells and embryos and possess a highly conserved (91% identical) amino acid sequence from humans to invertebrates. A homozygous Drosophila melanogaster strain mutant in the PP4 gene at 19C1-2 has been produced using P element mutagenesis. This strain, termed centrosomes minus microtubules (cmm), has reduced amounts of PP4 mRNA, approximately 25% of normal PP4 protein in early embryos and exhibits a semi-lethal phenotype with only 10% viability in certain conditions. Reversion mutagenesis shows that the phenotype is due to the presence of the P element in the PP4 mRNA. In early cmm embryos, nuclear divisions become asynchronous and large regions containing centrosomes with no well defined radiating microtubules are visible. In such areas, most nuclei arrest during mitosis with condensed DNA, and mitotic spindle microtubules are either absent, or aberrant and unconnected to the centrosome. A reduction in the staining of gamma-tubulin at centrosomes in cmm embryos suggests a conformational change or relocation of this protein, which is known to be essential for initiation of microtubule growth. These findings indicate that PP4 is required for nucleation, growth and/or stabilisation of microtubules at centrosomes/spindle pole bodies.

/pdf/protein-phosphatase-4-is-an-essential-enzyme-required-for-58t1fekbar.pdf

Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos

The sequences of two Drosophila and one rabbit protein phosphatase (PP) 1 catalytic subunits were determined from their cDNA. The sequence of Drosophila PP1 alpha 1 was deduced from a 2.2-kb cDNA purified from an embryonic cDNA library, while that for Drosophila PP1 beta was obtained from overlapping clones isolated from both a head cDNA library and an eye imaginal disc cDNA library. The gene for Drosophila PP1 alpha 1 is at 96A2-5 on chromosome 3 and encodes a protein of 327 amino acids with a calculated molecular mass of 37.3 kDa. The gene for Drosophila PP1 beta is localized at 9C1-2 on the X chromosome and encodes a protein of 330 amino acids with a predicted molecular mass of 37.8 kDa. PP1 alpha 1 shows 96% amino acid sequence identity to PP1 alpha 2 (302 amino acids), an isoform whose gene is located in the 87B6-12 region of chromosome 3 [Dombradi, V., Axton, J. M., Glover, D.M. Cohen, P.T.W. (1989) Eur. J. Biochem. 183, 603-610]. PP1 beta shows 85% identity to PP1 alpha 1 and PP1 alpha 2 over the 302 homologous amino acids. These results demonstrate that at least three genes are present in Drosophila that encode different isoforms of PP1. Drosophila PP1 alpha 1 and PP1 beta show 89% amino acid sequence identity to rabbit PP1 alpha (330 amino acids) [Cohen, P.T.W. (1988) FEBS Lett. 232, 17-23] and PP1 beta (327 amino acids), respectively, demonstrating that the structures of both isoforms are among the most conserved proteins known throughout the evolution of the animal kingdom. The presence of characteristic structural differences between PP1 alpha and PP1 beta, which have been preserved from insects to mammals, implies that the alpha and beta isoforms may have distinct biological functions.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1990.tb19464.x

Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2890%2981435-Q

Neil D. Brewis

Papers

Protein serine/threonine phosphatases; an expanding family

PPX, a novel protein serine/threonine phosphatase localized to centrosomes.

Protein phosphatase 4 is an essential enzyme required for organisation of microtubules at centrosomes in Drosophila embryos

Drosophila contains three genes that encode distinct isoforms of protein phosphatase 1

The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase I