Protein phosphatase 1 binding protein, R5

Details Protein phosphatase 1 binding protein, R5 Protein phosphatase 1 binding protein, R5

2000-01-10

2001-10-02

Eyler, Yvonne (Department: 1646)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

C435S069100, C435S183000, C536S023100

Reexamination Certificate

active

06297359

ABSTRACT:

FIELD OF THE INVENTION
The invention relates generally to newly identified polypeptides and polynucleotides, variants and derivatives of the polynucleotides and polypeptides; polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. In particular, the invention relates to novel polypeptides and polynucleotides related to a novel human protein related to the liver- and muscle-specific glycogen binding subunits of protein phosphatase 1. The novel protein is hereinafter called protein phosphatase 1 binding protein R5 or PPP1R5.
BACKGROUND OF THE INVENTION
Protein phosphatase 1 (PP1) participates in the regulation of a wide variety of cellular functions by reversible protein phosphorylation and is one of the major protein phosphatases dephosphorylating serine and threonine residues in eukaryotes. The ability of PP1 to regulate diverse functions resides in the capacity of PP1 to interact with a variety of regulatory subunits that may target PP1 to specific subcellular locations, modulate its substrate specificity, and allow its activity to be responsive to extracellular signals [Hubbard and Cohen,
Trends Biochem. Sci.,
18:172-177 (1993)].
A 126 kDa glycogen binding subunit (G
M
or PPP1R3) targets PP1 to glycogen particles and to the sarcoplasmic reticulum in striated muscle [Tang et al.,
J. Biol. Chem.,
266: 15782-15789 (1991); Chen et al.,
Diabetes,
43:1234-1241 (1994)]. The hormones insulin and adrenalin are thought to influence the activity of PP1 via the G
M
subunit. Phosphorylation of Ser-46 in human G
M
, response to insulin, enhances the rate at which at which PP1 dephosphorylates and activates glycogen synthase, causing an increase in glycogen synthesis [P. Dent et al,
Nature,
348:302-308 (1990)]. In contrast, phosphorylation of Ser-65 in human G
M
by protein kinase A in response to &bgr;-adrenergic agonists triggers dissociation of PP1 from G
M
, thus inhibiting PP1 from acting on glycogen synthase and phosphorylase and resulting in decreased glycogen synthesis and stimulation of glycogenolysis [Nakielny et al.,
Eur. J. Biochem.,
199: 713-722 (1991)].
A distinct 33 kDa glycogen binding subunit (G
L
or PPP1R4), which is only 23% identical to the N-terminal portion of G
M
, targets PP1 to glycogen in liver [Moorhead et al.,
FEBS Lett.,
362:101-105 (1994); Doherty et al.,
FEBS Lett.,
375:284-289 (1995)]. The binding of G
L
modulates the activity of PP1, enhancing the rate at which it dephosphorylates and activates glycogen synthase and suppressing the rate at which it inactivates phosphorylase. Hormonal regulation of the activity of PP1 in liver is not known to occur through the phosphorylation of G
L
. Instead, the hormone glucagon (acting via cyclic AMP and protein kinase A) and a-adrenergic agonists (acting via Ca
+2
) increase the levels of phosphorylase a, which to binds G
L
and potently inhibits PP1 at nanomolar concentrations. This inhibition is though to be allosteric, since the K
m
for phosphorylase as a substrate of PP1 is in the micromolar range. Insulin acts by lowering the level of cyclic AMP in liver, thereby decreasing the level of phosphorylase a and relieving the inhibition of PP1-G
L
complex. Glycogen synthesis in liver is also stimulated by glucose, which binds to phosphorylase a, increasing the rate at which it is dephosphorylated.
Several other targeting subunits of PP1 have now been identified in mammals and these include the myosin binding targeting complex (comprising an M
110
and M
21
subunit) of smooth muscle, which enhances the dephosphorylation of myosin light chains by PP1 and is involved in the relaxation of smooth muscle [D. Alessi et al,
Eur. J. Biochem.,
210:1023-1035 (1992); Y. H. Chen et al,
FEBS Lett.,
356:51-55 (1994)]. A distinct myosin targetting subunit of PP1 is present in striated muscles [P. Dent et al,
Eur. J. Biochem.,
210:1037-1044 (1992)]. A p53 binding protein (53BP2) [N. R. Helps et al,
FEBS Lett.,
377:295-300 (1995)], a nuclear protein NIPP-1 [A. Van Eynde et al.,
J. Biol. Chem.,
270:28068-28074 (1995)] and an RNA splicing factor PSF1 [K. Hirano et al,
FEBS Lett.,
389:191-194 (1996)] have been shown to bind to PP1. The retinoblastoma gene product [T. Durphee et al.,
Genes Dev.,
7:555-569 (1993)], ribosomal proteins L5 [K. Hirano et al.,
J. Biol. Chem.,
270:19786-19790 (1995)] and RIPP-1 [Buellens et al,
Eur. J. Biochem.,
239:183-189 (1996)] and a 110 kDa nuclear protein yet to be identified [I. Jagiello et al,
J. Biol. Chem.,
270:17257-17263 (1995)] are also reported to interact with PP1. The small cytosolic proteins, inhibitor-1, inhibitor-2 and DARPP-32 inhibit PP1 [P. Cohen,
Annu. Rev. Biochem.,
58:453-508 (1989)]. A complex between inhibitor-2 and PP1 has been isolated. More recently, inhibitor-2 has been shown to act like a molecular chaperon to fold PP1 into its native conformation [D. R. Alessi et al,
Eur. J. Biochem.,
213:1055-1066 (1993); C. MacKintosh et al,
FEBS. Lett.,
(1996) in press]. A number of distinct PP1 targeting subunits have also been identified in yeast [M. J. R. Stark,
Yeast
(1996) in press].
Sites on the glycogen and myofibrillar targeting subunits which bind to PP1 have been localized [D. F. Johnson et al.,
Eur. J. Biochem.,
239:317-325 (1996)] and a 13 residue peptide containing a RVXF motif common to many of the PP1 binding subunits has been crystallized as a complex with PP1 [M. Egloff et al.,
EMBO J.
(1997) submitted].
Known PP1 subunits play a role in the regulation of glycogen metabolism and thus novel PP1 subunit proteins, agonists or antagonists thereof are anticipated to be beneficial in many diseases which involve resistance to the action of insulin on glycogen synthesis. Such disorders include, without limitation, diabetes mellitus, obesity, essential hypertension, dyslipidaemia and premature atherosclerosis, among others.
There, thus, exists a need in the art for a variety of PP1 binding proteins, antagonists and agonists thereof, as well as compositions and methods for the use of same.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides polypeptides, inter alia, that have been identified as novel PP1 binding subunit polypeptides of human origin, as well as biologically active and diagnostically or therapeutically useful fragments, variants, analogs and derivatives thereof, variants and derivatives of the fragments, and analogs of the foregoing. These polypeptides have been identified as PP1 binding subunit polypeptides, e.g., by homology between the amino acid sequence set out in
FIGS. 1A-1B
and known amino acid sequences of other proteins such as G
M
and G
L
(see FIG.
4
). In one embodiment, the present invention provides a novel human PP1 binding subunit which contains the PP1 binding motif, modulates the specificity of PP1 and is related to G
L
, but has a much wider tissue distribution. This binding subunit is referred to as PPP1R5 in accordance with the human genome nomenclature [P. T. W. Cohen,
Adv. Prot. Phosphatases,
8:371-376 (1996)].
In another aspect of the present invention, there are provided non-naturally occurring synthetic, isolated and/or recombinant PPP1R5 polypeptides, fragments, consensus fragments and/or sequences having conservative amino acid substitutions of the PPP1R5 of the present invention. These polypeptides may bind PP1, or may also modulate, quantitatively or qualitatively, PPP1R5 ligand binding.
In another aspect, the present invention provides synthetic, isolated or recombinant polypeptides which are designed to inhibit or mimic various PPP1R5 proteins or fragments thereof.
In another aspect of the invention, there are provided isolated nucleic acid molecules encoding PPP1R5 polypeptides, particularly human PPP1R5. Such molecules include polynucleotides, mRNAs, DNAs, cD

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