Rapid Identification of Protein Phosphatase 1-binding Proteins by Mixed Peptide Sequencing and Data Base Searching

Abstract

Microcystin-affinity chromatography was used to purify 15 protein phosphatase 1 (PP1)-binding proteins from the myofibrillar fraction of rabbit skeletal muscle. To reduce the time and amount of material required to identify these proteins, proteome analysis by mixed peptide sequencing was developed. Proteins are resolved by SDS-polyacrylamide gel electrophoresis, electroblotted to polyvinylidene fluoride membrane, and stained. Bands are sliced from the membrane, cleaved briefly with CnBr, and applied without further purification to an automated Edman sequencer. The mixed peptide sequences generated are sorted and matched against the GenBank using two new programs, FASTF and TFASTF. This technology offers a simple alternative to mass spectrometry for the subpicomolar identification of proteins in polyacrylamide gels. Using this technology, all 15 proteins recovered in PP-1C affinity chromatography were sequenced. One of the proteins, PP-1bp55, was homologous to human myosin phosphatase, MYPT2. A second, PP-1bp80, identified in the EST data bases, contained a putative PP-1C binding site and a nucleotide binding motif. Further affinity purification over ATP-Sepharose isolated PP-1bp80 in a quaternary complex with PP-1C and two other proteins, PP-1bp29 and human p20. Recombinant PP-1bp80 also bound PP-1C and suppressed its activity toward a variety of substrates, suggesting that the protein is a novel regulatory subunit of PP-1.Protein phosphatase 1 (PP-1, 1 EC 3.13.16) dephosphorylates serine and threonine residues on proteins that control a diverse range of cellular processes from metabolism and muscle contraction to the cell cycle and gene expression (1). The involvement of PP-1 in all of these events raises the question as to how all of these processes can be regulated independently of one another? This question is even more apparent when one considers that the catalytic subunit of PP-1 (PP-1C) is expressed at micromolar concentrations in cells and shares 49% sequence homology within its catalytic core with at least 3 other highly expressed phosphatases, PP-2A, PP-2B, and PP-5 (2-5). The key to this paradox is the finding that the functions of PP-1 are closely linked to its subcellular localization with regulatory targeting subunits that confer substrate specificity (1). At the time of writing, 10 regulatory subunits have been sequenced at the protein and DNA level in mammalian tissues, and at least 10 others identified in Saccharomyces cerevisiae (6). Five of the mammalian subunits potently inhibit the enzyme's activity toward all substrates, whereas the others target the activity of PP-1C toward myosin, glycogen synthase, p53, or the nucleus (6). The small number of PP-1 regulatory proteins discovered thus far is unlikely to account for all the intracellular actions of the phosphatase, suggesting that many others must exist.One of the mechanisms by which PP-1C recognizes its regulatory subunits was recently characterized (6). Amino acid sequence alignments of nine of the mammalian regulatory subunits identifie...

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase

“…As described above, the ZIP-like kinase is closely related to ZIPK that in turn is related to the death-associated protein kinase (34), and the latter two are involved in the mediation of apoptosis (24,34). Whether the ZIP-like kinase is involved in similar cell functions is not known and there are no data implicating MYPT1 in cell death.…”

“…Fractions containing SMPP-1M kinase activity were separated by SDS͞PAGE and electroblotted to polyvinyl membrane. The transferred proteins were stained with Amido Black and identified by mixed peptide sequences as described (24).…”

A Strategy for the Rapid Identification of Phosphorylation Sites in the Phosphoproteome

“…The treated piece of PVM was placed in an Applied Biosystem 494 protein sequencer, and 8 -18 cycles of pulsed liquid chemistry were carried out. The mixed peptide sequences generated were sorted and matched against the yeast protein databases by the FASTF algorithm (9).…”

Protein phosphatase 1α-mediated stimulation of apoptosis is associated with dephosphorylation of the retinoblastoma protein

“…The assignment of speci®c roles in various cellular processes to PP1 is complicated by the fact that mammalian PP1 comprises four isozymes (1a, 1d, 1g1, 1g2) which can bind to many non-substrate cellular proteins functioning as regulatory subunits (Damer et al, 1998). These subunits modify PP1's activity or target it to particular locales and substrates.…”