Norbert Berndt

Constitutively active protein phosphatase 1α causes Rb-dependent G1 arrest in human cancer cells

BACKGROUND The retinoblastoma protein (Rb) needs to be phosphorylated by cyclin-dependent kinases (CDKs) before mammalian cells can enter the S phase of the cell cycle. As protein phosphatase 1 (PP1) activates Rb and is itself a target for inhibitory phosphorylation by CDKs in vitro, we asked whether any effects of PP1 on cell cycle progression depend on its phosphorylation and are mediated through Rb. RESULTS Using electrotransfer of recombinant protein into Rb-positive and Rb-negative cells, we have compared the effects of a wild-type PP1 catalytic subunit, PP1alpha, and a constitutively active mutant of this subunit (PP1alphaT320A) on G1 progression, proliferation rates, and cell viability. In treated cells, PP1alpha levels were elevated 6-16-fold and remained stable for at least 48 hours. In Rb-positive cells, PP1alphaT320A, but not PP1alpha, caused cell cycle arrest in late G1, which was associated with a lack of Rb phosphorylation. In Rb-negative cells, neither wild-type nor mutant phosphatase caused any change in cell cycle progression. Increased cell death was observed in both Rb-positive and Rb-negative cells, however, upon introduction of excess PP1alpha. CONCLUSIONS The difference between the effects of wild-type and mutant forms of PP1alpha suggests that PP1alpha has the potential to arrest cell growth in G1 unless it is inactivated by periodic phosphorylation at Thr320, presumably by CDKs that regulate passage through the G1-S cell cycle transition. Together, the effects in both cell types suggest that PP1alpha requires functional Rb to induce growth arrest, and that possibly another pool of PP1alpha induces cell death. This identifies PP1 as a potential target for therapeutic anti-proliferative strategies.

Inhibitory Phosphorylation of PP1α Catalytic Subunit during the G1/S Transition

We have shown earlier that, in cells expressing the retinoblastoma protein (pRB), a protein phosphatase (PP) 1α mutant (T320A) resistant to inhibitory phosphorylation by cyclin-dependent kinases (Cdks) causes G1arrest. In this study, we examined the cell cycle-dependent phosphorylation of PP1α in vivo using three different antibodies. PP1α was phosphorylated at Thr-320 during M-phase and again in late G1- through early S-phase. Inhibition of Cdk2 led to a small increase in PP1 activity and also prevented PP1α phosphorylation. In vitro, PP1α was a substrate for Cdk2 but not Cdk4. In pRB-deficient cells, phosphorylation of PP1α occurred in M-phase but not at G1/S. G1/S phosphorylation was at least partially restored after reintroduction of pRB into these cells. Consistent with this result, PP1α phosphorylated at Thr-320 co-precipitated with pRB during G1/S but was found in extracts immunodepleted of pRB in M-phase. In conjunction with earlier studies, these results indicate that PP1α may control pRB function throughout the cell cycle. In addition, our new results suggest that different subpopulations of PP1α regulate the G1/S and G2/M transitions and that PP1α complexed to pRB requires inhibitory phosphorylation by G1-specific Cdks in order to prevent untimely reactivation of pRB and permit transition from G1- to S-phase and/or complete S-phase.

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

Protein phosphatase 1 (PP1) plays important roles in many different aspects of cellular activities including cell cycle control. One important function of PP1 is to activate the retinoblastoma protein pRB. Here we show that pRB is one of PP1s downstream targets during apoptosis. When HL-60 cells synchronized at the G1/S boundary were treated with pro-apoptotic cytosine arabinoside (araC), PP1α protein increased twofold and PP1 activity about 30% within 1 h. This was followed by pRB dephosphorylation, pRB cleavage by caspases, DNA fragmentation, the appearance of cells with <2n DNA content and finally, dying and dead cells. In vitro, pRB was protected from caspase-3 digestion by prior Cdk-mediated phosphorylation, whereas PP1α converted phospho-pRB into an efficient substrate for caspase-3. Introduction of active PP1α into HL-60 cells by electroporation was sufficient to induce characteristics of apoptosis. Similarly, araC-resistant cells, normally unable to die in response to araC, initiated apoptosis when electroporated with active PP1α. This was also accompanied by pRB cleavage. In contrast, introduction of inhibitor-2 delayed the onset of araC-induced apoptosis, whereas concomitant introduction of PP1α and inhibitor-2 completely prevented PP1α-induced apoptosis. These results suggest that dephosphorylation of key proteins by PP1α may be crucial for the initiation of apoptosis and further support the concept of PP1 serving as a potential target for anti-cancer therapy.

Protein phosphatase 1α activity prevents oncogenic transformation

Cyclin‐dependent kinase 2 (Cdk2) phosphorylates Thr320 of protein phosphatase 1α (PP1α) in late G1, thereby inhibiting its activity. Phosphorylation‐resistant PP1αT320A, acting as a constitutively active (CA) mutant, causes a late G1 arrest by preventing the phosphorylation and inactivation of the retinoblastoma protein (pRb). Both PP1α‐mediated G1 arrest and PP1α phosphorylation in late G1 require the presence of pRb, indicating that PP1α is a crucial regulator of the pRb pathway, which is almost invariably mutated in human cancer. These findings prompted us to investigate whether PP1α interferes with oncogenic transformation. The ability of NIH 3T3 cells to form foci after transformation with ras/cyclin D1 was significantly inhibited by co‐transfection with PP1αT320A, but not PP1α. Likewise, cells expressing PP1αT320A or PP1αT320A fused to green fluorescent protein (GFP) were unable to form colonies in soft agar, regardless of whether PP1α constructs were co‐transfected with ras/cyclin D1 or transfected into stably transformed cells. Overexpressed wild‐type (Wt) PP1α and GFP‐PP1α were phosphorylated in Thr320, most likely explaining its lack of effect. Expression of GFP‐PP1αT320A was associated with caspase‐cleaved pRb in Western blots (WB) and morphological signs of cell death. These findings demonstrate that PP1α activity can override oncogenic signaling by causing cell‐cycle arrest and/or apoptosis rather than restoring contact inhibition or anchorage dependence.

Protein phosphatase 1α is required for murine lung growth and morphogenesis

Protein phosphatase 1 (PP1) plays important roles in cell cycle control and apoptosis, two processes that impinge on morphogenesis and differentiation. Following the precedent set by other molecules regulating the cell cycle and apoptosis, we hypothesized that PP1 may have context‐specific roles in development. Therefore, we have studied the spatial and temporal expression of PP1α during murine lung development and determined the consequences of loss of PP1α function on branching morphogenesis. By using an immunohistochemical approach, we show here that PP1α was expressed throughout the epithelium and mesenchyme upon the emergence of the lung primordium on embryonic day 10, with immunostaining exclusively extranuclear. During the late pseudoglandular stage, PP1α was predominantly expressed in the distal lung epithelium, whereas the mesenchyme contained very little or no PP1α protein. Peri‐ and postnatally, PP1α immunostaining was mostly nuclear in apparently differentiated cells, as judged by colocalization with well‐known markers for lung differentiation. Exposure of fetal lung explants to antisense oligodeoxynucleotides against PP1α, resulted in decreased overall size of the cultured lung, a defect in forming new airways, lack of expression of surfactant protein C, and histologic signs of poor differentiation. These data suggest that PP1α is required for branching morphogenesis and differentiation. Developmental Dynamics 229:791–801, 2004.

Serine/threonine-specific protein phosphatases and cancer

In a multicellular organism, the cell number depends on the balance between cell proliferation and programmed cell death or apoptosis. Human cancer is a disease that involves unrestrained cell cycle progression and/or a failure to undergo apoptosis. While we have identified the essential pathways controlling the cell number, we have realised that these two processes are regulated by protein phosphorylation/dephosphorylation. Considerable effort is now being devoted towards testing and exploiting molecular targets that can improve the mostly frustrating results of present chemotherapy. Given that our understanding of protein phosphorylation is far better than that of protein dephosphorylation, it is not surprising that most of the work is currently focused on select protein kinases, their regulators and substrates. This review will discuss recent advances in the characterisation of protein phosphatases, which play a role in controlling the cell number and, at the same time, attempt to identify important areas for future research. While protein kinases in general function as positive regulators of cell cycle progression and cell survival, most, but not all, protein phosphatases have been shown to have the opposite effect. Several phosphatases decelerate or block cell cycle progression and/or facilitate cell death. Collectively, these findings raise the possibility that protein phosphatases are novel targets for significantly improved therapy of cancer. Specific approaches to exploit this potential will be discussed.