The activities of PP1 (protein phosphatase 1), a principal cellular phosphatase that reverses serine/threonine protein phosphorylation, can be altered by inhibitors whose activities are themselves regulated by phosphorylation. We now describe a novel PKC (protein kinase C)-dependent PP1 inhibitor, namely GBPI (gut and brain phosphatase inhibitor). The shorter mRNA that encodes this protein, GBPI-1, is expressed in brain, stomach, small intestine, colon and kidney, whereas a longer GBPI-2 splice variant mRNA is found in testis. Human GBPI-1 mRNA encodes a 145-amino-acid, 16.5 kDa protein with pI 7.92. GBPI contains a consensus PP1-binding motif at residues 21–25 and consensus sites for phosphorylation by enzymes, including PKC, PKA (protein kinase A or cAMP-dependent protein kinase) and casein kinase II. Recombinant GBPI-1-fusion protein inhibits PP1 activity with IC50=3 nM after phosphorylation by PKC. Phospho-GBPI can even enhance PP2A activity by >50% at submicromolar concentrations. Non-phosphorylated GBPI-1 is inactive in both assays. Each of the mutations in amino acids located in potential PP1-binding sequences, K21E+K22E and W25A, decrease the ability of GBPI-1 to inhibit PP1. Mutations in the potential PKC phosphoacceptor site T58E also dramatically decrease the ability of GBPI-1 to inhibit PP1. Interestingly, when PKC-phosphorylated GBPI-1 is further phosphorylated by PKA, it no longer inhibits PP1. Thus, GBPI-1 is well positioned to integrate PKC and PKA modulation of PP1 to regulate differentially protein phosphorylation patterns in brain and gut. GBPI, its closest family member CPI (PKC-potentiated PP1 inhibitor) and two other family members, kinase-enhanced phosphatase inhibitor and phosphatase holoenzyme inhibitor, probably modulate integrated control of protein phosphorylation states in these and other tissues.
- cell signalling
- PKA (protein kinase A)
- PKC (protein kinase C)
- PP1 (protein phosphatase 1)
The nucleotide sequence data reported have been submitted to the GenBank® Nucleotide Sequence Databases under the following accession numbers: human GBPI-1 cDNA, AY050671; mouse GBPI-1 cDNA, AF408400; mouse GBPI-2, AY179331; rat GBPI-1 cDNA, AY122322; human CPI cDNA, AY050670; mouse CPI cDNA, AY050672; rat CPI cDNA, AY050673; rat PHI-1 cDNA, AY122323; pig PHI-1 cDNA, AY122324; and Drosophila melanogaster KEPI cDNA, AY050669.
Abbreviations used: PKA, protein kinase A; PKC, protein kinase C; DARPP, dopamine- and cAMP-regulated phosphoprotein; PP1, protein phosphatase 1; CPI, PKC-potentiated PP1 inhibitor; GBPI, gut and brain phosphatase inhibitor; GPCR, G-protein-coupled receptor; GST, glutathione S-transferase; KEPI, kinase-enhanced phosphatase inhibitor; NIPP1, nuclear inhibitor of PP1; ORF, open reading frame; PHI, phosphatase holoenzyme inhibitor; RT, reverse transcriptase; THOP, testis homologue of PHI; for brevity, the one-letter system for amino acids has been used: e.g. E28 is Glu28.
- The Biochemical Society, London ©2004