Regulatory subunits interactions

Phosphorylation is one of the most prevalent processes used in regulating all eukaryotic

cellular proteins and in some studies, it said that 70% of the cells are regulated by this process.

However, it should be noted that the phosphorylation process itself is regulated by the intricate

action of phosphates and kinases on serine/threonine or tyrosine residues [11]. In the human

genome, more than 420 genes encode for threonine/serine kinases which are approximately

98.2% of entire phosphorylation actions, only about 40 genes encode for threonine/serine

phosphates (PSP). The largest family of the PSP is the Phosphate Protein Phosphate (PPP) and it

consists of seven members (PP1-PP7), with the PP1 and the PP2A being the two most prevalent

enzymes in numerous cell types. However, dephosphorylation of p-Thr, p-Ser and p-Tyr can be

done by dual-specificity phosphates (DUSPs) [8]. The most characterized threonine/serine

phosphate is the PP1 (protein phosphates 1), and the human genome has three distinct genes

which encode four various PP1’s catalytic subunits and they include the splice variants PP1γ1

and PP1γ2, PP1β/δ, and PP1α.

Concerning regulation, the heat-stable protein inhibitor-1 and inhibitor-2 are responsible

for regulating the catalytic subunits of PP1. The homologue for inhibitor-1 is the dopamine- and

cAMP-regulated phosphoprotein, Mr 32,000 (DARPP-32). For the inhibition of the PP1,

phosphorylation of the DARPP-32 at T-32 or inhibitor-1 at 34 as required [5]. On the contrary, the

inhibition can table place via the interaction of the PP1 catalytic subunits with the inhibitor-2,

which contributes to the formation of an inactive complex known as Mg-ATP-dependent

phosphate which leaders to the inhibition [11]. It should be noted that PP1 also interacts with

numerous regulatory subunits which are responsible for targeting the catalytic subunits to

specific subcellular compartments. Some of these targeting subunits include spinophilin and
neural (spine-targeting proteins), PP1 nuclear targeting subunit, glycogen-targeting subunit, the

nuclear targeting protein and myofibrillar-targeting subunit.

The PP1 interacts with targeting subunits and inhbitor1/DARPP-32 through a popular

docking motif which is composed of hydrophobic residues and either one or more amino acids

(aa) which are separated by a variable aa. The hydrophobic channel of the PP1 located opposite

the active site interacts extensively with the docking motif (RRVSF). β-strands, such as the β12,

β13, and β14 are some of the residues which form the hydrophobic channel. Therefore,

inhibitor.1/DARPP-32 in addition to the docking motif makes more contact with the above-

named β-strands such that the phosphor-(T-35/T-34) occupies or comes close to the active site of

PP1 [7]. On the contrary, inhibitor-2 has a special N-terminal motif that integrates with the region

of pp1 that is slightly removed from the discussed hydrophobic channels and hence it does not

have a canonical RRVSF docking motif. The β12, β13, and β14 strands have a significant

function which is highly unique interactions of targeting subunits and protein inhibitors with PP1
[2]
. The chimeric phosphatase is made insensitive to inhibitor-2 and inhibitor-1 through the

replacement of PP1 terminal C, involving the above three named strands with a correspondent

area of a phosphatase which is related to PP1 structurally (PP2).

A series of various proteins determine the subcellular localization of PP1 which is

currently considered essential subunits of this enzyme. For instance, PP1 is targeted to myofibrils

in smooth muscles by the M subunit. Also, in the brain, the isoforms of PP1 demonstrate a

region-specific expression. Some forms, such as the PP1γ1 and PP1α are more enriched in the

dendritic spines of some neurons and therefore they reflect the essential function played by the

PP1 enzyme in the synaptic transmission. The protein spinophilin is probably the subunit that is

responsible for the above-described dendritic localization [12]. The nucleus of mammalian cells
also a huge amount of PP1, which is associated with the crucial role of dephosphorylation of

transcription factors. Therefore, this is the reason behind which the PP1 protein is commonly

known for its role in regulating the gene transcription in the cell nucleus.

The phosphatase 1 nuclear targeting subunit (PNUTS) is the PP1 protein that is

responsible for nuclear targeting. It should be noted that PP1 plays an essential role in the

neurons because it dephosphorylates numerous neuronal phosphoproteins and therefore the

actions of various proteins Thr/Ser kinases are antagonized. For instance, PP1-catalyzed protein

dephosphorylation has been shown to be a critical factor of the signalling pathways underlying

synaptic plasticity long-lasting forms. it is worth noting that PP1 is involved in the

dephosphorylation of a variety of substrate proteins. The PP1 catalytic subunits can formulate

with more than 50 targeting/regulating proteins in a mutually exclusive manner. The PP1

interaction with these proteins impacts its activities], subcellular localization, and substrate

specificity[10]. Although for some substrates has various binding sites which are best suited for

the PP1 enzyme, localization of the PP1 is essential because some substrate only bind and get

dephosphorylated with the aid of other catalysts such as the PIPs. Also, localization

determination is essential as some cells or tissues require PP1 enzyme given that the

dephosphorylation process is essential to how they function.

Concerning substrate specificity of the catalytic subunits of protein phosphatase 1, it

should be noted that the PP1 catalytic core differs from that of other PPP superfamily due to the

difference in affinity for ligands brought about by the solvent-exposed loops. Some acids

residues conspicuously surround the catalytic sites of the PP1 [8]. The above feature is the

rationale as to why the β-subunit of phosphorylase kinase is faster dephosphorylated compared to

much acidic α-subunit. The above ability is essential in biochemistry because it enhances
researchers and technicians to differentiate PP1 from other protein serine/threonine phosphates.

The other property of PPI is that its ability to dephosphorylate short peptides formed base on its

physiological substrates is low which shows that unlike numerous protein kinases,

phosphorylated residues surrounded by consensus sequence is not recognized by PP1.

The surface grooves of docking motifs remote from the active site for PP1 determine the

efficiency of substrate binding. The substrate specificity of the PP1 under controlled buffer

condition is broad [10]. As such, the PP1 expressed by the bacterium in mammals can serve as a

protein tyrosine phosphate and which can be used in the dephosphorylation of small molecules

like p-nitrophenylphosphatase. However, these atypical substrates cannot be acted upon by

purified catalytic subunits from mammalian tissues. The PP1 enzyme is more selective which

could be due to the metals that are contained in the active sites such as zinc and iron ions are

different from the ones contained in the bacterially expressed sites (Mn2+).

There are two groups of PP1 and the classification of the physiological substrate of PP1

is done based on the affinity for the catalytic subunit. PP1 substrates have high-affinity docking

sites which with phosphatase form heterodimeric complexes even when they are

dephosphorylated. However, some of the interactions between substrates such as glycogen

phosphorylase and catalytic subunits are weak which can be evidenced by the nearly complete

inhibition of their dephosphorylation by their inability to form stable complexes with PP1 and

the physiological salt concentrations (Heroes et al., 2013). Therefore, successful in vivo

dephosphorylation of substrates like glycogen phosphorylase requires PIPs which increases the

local substrate concentration by tethering the phosphate to a substrate containing compartments

or by providing additional substrate docking sites. Therefore, the selection of substrates by the
PP1 is dependent on the subcellular targeting subunits and phosphate docking motif that together

contain the substrate-specifying and -targeting toolkit of PP1.

Functional and regulatory effects of PP1-binding proteins

The PSF factor is a 100 kDa protein that has been associated with multiple steps in

splicing. PSF consist of a rich domain of proline/glutamine, a C-terminal region with two nuclear

localization signals, an N-terminal glycine-rich domain, and two RNA recognition motifs

(RRMs). In addition to splicing, PSF has been associated with other numerous nucleic activities

such as acting as a regulating factor during transcription in cases like regulation of growth factor

stimulating expression, inducing silencing, and as the complex protein responsible for activating

the CYP17 promoter responding to the cAMP stimulation [1]. Regardless of the addition of the

pre-mRNA, all the interactions of HSF with U snRNPs co-IP when nuclear extracts, HeLa, are

adjusted to splicing conditions. The formation of the large PSF complex of multi-snRNP in

nuclear extracts is similar to the conditions which are usually utilized for in-vitro splicing. After

sedimentation, the smaller PSF-containing complexes all the five snRNPs which resemble

mature spliceosomes where are the largest complexes usually have non-specific aggregates [3]. It

is crucial to note that the regulation of the gene transcription process is integral to the production

of new proteins in the cells and hence the cells can hardly function without the presence of the

splicing factor PSF.

As highlighted above, PSF participates in various cellular activities including apoptosis,

RNA transport, and the repair of DNA. In conjunction with the PSPC1 and the p54nrb, it is

associated with the Drosophila behaviour/human splicing (DBHS) which is a well-conversed

multifunctional protein family which is used in the formation of obligated hetero- and

homodimers [9]. PSF is highly related to the role of ensuring the viability of cells. Although the
precise process by which the PSF is needed for cell survival and growth is not entirely apparent,

there is evidence which associates it with the regulation of apoptosis [3]. The above argument is

because numerous transcriptions encoding for proteins used in apoptosis pathway supposed to

undergo alternative splicing which contributes to protein expression with opposite roles either

anti- or proapoptotic. Variations in alternative splicing patterns are usually exhibited under the

selective pressure of cancer patient treatment or in conjunction with acquired chemoresistance.

For instance, in the case of breast cancer cells, more than 600 transcripts from alternative

splicing are induced in breast cancer treatment using cisplatin [9]. Among the 600 transcripts,

some are involved apoptosis-like the BCL2L1, caspase-8, P73, Diablo, and Fas, are present in a

cancer patient undergoing the treatment and hence this indicates that the death of the cells during

cancer is partly regulated by the splicing factor PSF.

As seen above, splicing factor PSF is very essential in gene transcription but in the

following section, we are going to see that the ribosome protein of the PP1 proteins is also very

essential in gene transcription. To better comprehend the initial point for gene regulation and the

construction of a gene regulatory network, one has to understand the ribosome protein. The basic

function of ribosomes in all cells is to synthesize proteins and it is composed of 4 ribosomal

RNAs (rRNA) and approximately 80 ribosome proteins especially in mammals [6]. The ribosome

proteins in higher eukaryotes are scattered through the entire genome and still, these genes are

expressed even without operon structure. However, the 4 rRNAs play a very essential role in the

transcription because they are involved in the formation of a core of a cell’s ribosomes which is

the structure in which the synthesis of protein takes place. Therefore, transcription in eukaryotes

is entirely dependent on the efficiency of the rRNAs which are components of ribosome proteins.

Therefore, it can be concluded that the ribosome proteins are involved in the regulation of
transcription since some of its components are involved in the pathway used in protein synthesis

in cells.

Ribosome proteins are integral in maintaining cell survival and homeostasis through the

coordination of protein biosynthesis as clarified above. However, some ribosomal proteins

posses a secondary role that is independent of their involvement in the synthesis of proteins.

These secondary functions exhibited by these ribosome proteins are acting as regulators of cell

proliferation and in some cases as inducers of apoptosis or cell death [4]. More specifically, the

expression of L13a ribosome protein which is found in humans is known to induce apoptosis that

is done in the G2/M phase of the cell cycle by arresting the cell growth. Besides, the expression

inhibition of L13a causes apoptosis in targeted cells, which suggests that this protein plays an

integral part in the survival of cells. Another ribosome protein L7 is also associated with

apoptosis as it is involved in arresting the G1 phase of cell growth [6]. Therefore, these two PP1

binding proteins (ribosome protein and splicing factor PSF) have similar functional ad regulatory

effects which are mainly apoptosis and gene transcription. Rather, they are involved in the

pathways of the above impacts although their specific roles are not intertwined.
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