Cloning and identification of MYPT3: a prenylatable myosin targetting subunit of protein phosphatase 1.
To identify novel protein phosphatase 1 (PP1)-interacting proteins, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the catalytic subunit of PP1 as bait. In the present work, the isolation, identification and initial biochemical characterization of a novel PP1-interacting protein, MYPT3, which is homologous with the myosin phosphatase targetting subunit (MYPT) family, is described. MYPT3 aligns >99% with a region of mouse genomic DNA clone RP23-156P23 and localizes to chromosome 15, between markers at 44.1-46.5 cM, as demonstrated by radiation hybrid mapping. The gene consists of ten exons that encode for a 524-amino acid sequence with a predicted molecular mass of 57529 Da. The N-terminal region of MYPT3 consists of a consensus PP1-binding site and multiple ankyrin repeats. MYPT3 is distinguished from related approximately 110-130 kDa MYPT subunits by its molecular mass of 58 kDa, and a unique C-terminal region that contains several potential signalling motifs and a CaaX prenylation site. We have shown that affinity-purified glutathione S-transferase (GST)-MYPT3 is prenylated by purified recombinant farnesyltransferase in vitro. Endogenous PP1 from 3T3-L1 lysates specifically interacts with MYPT3. Additionally, purified PP1 activity was inhibited by GST-MYPT3 toward phosphorylase a, myosin light chain and myosin substrate in vitro. Overall, our findings identify a novel prenylatable subunit of PP1 that defines a new subfamily of MYPT.
Skinner
J A
JA
Department of Cell Biology, Pfizer Global Research and Development, Ann Arbor Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105, USA.
Saltiel
A R
AR
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D051379:Mice; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017368:Protein Prenylation; D021382:Protein Sorting Signals; D021122:Protein Subunits; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D020798:Two-Hybrid System Techniques
D018994:Myosin Light Chains; D021382:Protein Sorting Signals; D021122:Protein Subunits; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D009218:Myosins