Vav1 is a guanine nucleotide-exchange factor (GEF) that regulates MAPK pathway signaling. Its physiological expression is restricted to hematopoietic systems  and up-regulated by ATRA in APL-derived promyelocytes . In malignant promyelocytes, Vav1 interacts with both cytoplasmic and nuclear signaling molecules and participates in interconnected networks regulating the different aspects of ATRA-induced differentiation of APL-derived cells . ATRA drives Vav1 expression and increases association of Vav1 and c-Raf, putatively promoting sustained MAPK pathway activation, cell cycle arrest and differentiation . Vav has been found to be needed for myelopoiesis in knockout mice [31, 32]. In ATRA-induced differentiation of leukemia cells, Vav1 also interacts with PU.1, recruiting it to the promoter to transcriptionally activate expression of the CD11b differentiation marker . IRF is the transcription factor known to be the primary effector of interferon action. It is known to collaborate with ATRA . Like Vav1, IRF-1 also enhanced Raf/Mek/Erk activation and promotes ATRA-induced differentiation and cell cycle arrest .
In the wt parental cells, ATRA and roscovitine treatments reveal two main clusters determined by absence or presence of ATRA. Each of these resolves into cells without roscovitine or with roscovitine. The main determinant of variance is hence ATRA which is modified by roscovitine, so biologically roscovitine is just a modifier of a cellular response to ATRA, which is the main driver.
The ensemble of measured regulatory molecules responding to treatment segregates into two main clusters, cell cycle regulators and cell signaling differentiation regulators. Confirming the fidelity of the analysis to known cell cycle biology, the cell cycle regulators show the anticipated relationships, except for one revealing detail. In this cell cycle cluster, pS608 RB is coupled to CDK2, which is known to phosphorylate RB, and Cyclin E1 is coupled to both of these, which reflects the classical Cyclin E1 regulation of CDK2 . Somewhat surprisingly the putative canonical inhibitory and enhancing phosphorylation events, pY15 and pT160, of CDK2 are coupled and co-regulated (Figure 8A). Significantly the CDKI, p27 Kip1, is not in this cluster, although it is a classical inhibitor of CDK2 and mediates cell cycle arrest as is occurring under the influence of ATRA and ATRA plus roscovitine. The signaling regulators segregate into three discernible clusters that are followers that co-vary together with pS259-c-Raf as their driver. Notably pS259-c-Raf Raf is the driver for these three signaling subclusters, consistent with the postulated regulatory significance of pS259 Raf and its nuclear translocation in ATRA-induced differentiation . Of the three signaling molecule clusters, one contains Fgr and pY416 Src, which in this process we biochemically established as linked , another contains the Lyn SFK, and the third includes pS289/296/301-c-Raf coupled to p27 Kip1 (Figure 8A). So, the p27 Kip1 surprisingly appears in the group of signaling molecules covarying with c-Raf/phospho-c-Raf. Interestingly each of these signaling subclusters also has an entity that goes from essentially not expressed in untreated cells to clearly expressed in ATRA treated cells, namely Fgr, IRF-1, and p27 Kip1. And addition of roscovitine enhances these up-regulations. The coupling of the p27 Kip1 CDKI with a putative major signaling regulator of differentiation suggests how signaling driving differentiation is coupled to driving cell cycle arrest. Indeed, the p27 Kip1 gene promoter is Sp1 regulated  where the Sp1 transcription factor is a classical responder to Raf/Mek/Erk axis MAPK pathway signaling . p27 kip1 may thus be a molecular link connecting differentiation signaling to cell cycle arrest. Principal components analysis (PCA) revealed essentially only one principal component (Figure 8B) where pS289/296/301 c-Raf, Lyn, c-Cbl, and IRF1 were coupled as one major contributor and p27 Kip1, Cyclin E1, Cdk2, pY15 Cdk2, pT160 Cdk2, RB and pS608 RB were coupled as the other major contributor. The cell cycle regulators including p27 Kip1 appear coupled as a group (Figure 8C).
In the present work, we reported that roscovitine collaborates with ATRA to cause nuclear enrichment of proteins known to drive differentiation and cell cycle arrest of the t(15;17)-negative HL-60 human myeloblastic leukemia model. An ensemble of traditionally regarded cytosolic signaling molecules was unexpectedly found in the nucleus where their expression or phosphorylation state was regulated by ATRA. Roscovitine was found to target these and enhance effects of ATRA. One of these molecules was c-Raf. ATRA is known to cause its enrichment in the nucleus where it functions in a non-canonical signaling role to target transcription factors that drive differentiation . In the current study, we found that roscovitine enhanced the ATRA effect. HL-60 cells co-treated with ATRA and roscovitine showed increased nuclear c-Raf levels. c-Raf function in various contexts is controlled by site-specific phosphorylation that controls its binding to various other proteins . How roscovitine caused activation of kinases other than CDKs is unknown. In the current study, we see that roscovitine enhanced ATRA-induced nuclear c-Raf phosphorylation at S259 and S289/296/301, which are known to be associated with differentiation . Although nuclear c-Raf phosphorylated at S621 is implicated in myeloid cell differentiation , the potential roles of other sites (S259 and S289/296/301) remain to be determined. In the nucleus, the RB tumor suppressor protein is known to be central in cell cycle regulation and by inference differentiation. During the cell cycle progression of untreated HL-60 cells, RB is in the hyper-phosphorylated state but begins to be hypo-phosphorylated in late G2 in ATRA-treated cells . Hypo-phosphorylated RB is only detectable in cells undergoing differentiation . We found that the c-Raf in the nucleus interacted with RB and specifically with pS608 RB. pS608 is the hinge region phosphorylation that controls E2F binding and cell cycle progression. The ATRA-induced loss of pS608 RB with cell cycle arrest is associated with less RB and specifically less pS608 RB bound to Raf, even as the amount of nuclear c-Raf increases. Roscovitine promoted the loss of c-Raf bound with RB. Hence cell cycle arrest with loss of pS608 RB liberated c-Raf from RB and resulted in more c-Raf availability to stoichiometrically favor targets such as transcription factors that drive differentiation. This provides a heuristic mechanistic rationalization coupling cell cycle arrest and differentiation through the availability of non-RB-sequestered Raf.
We then explored Vav1 expression and found that roscovitine increases ATRA-induced nuclear enrichment of Vav1. Vav1, a guanine nucleotide exchange factor, plays a central role in the activation of MAPK signaling cascade  and is associated with downstream expression of the differentiation markers CD38 and CD11b. Brugnoli etal.  reported that the ATRA-induced expression of Vav1 recruits PU.1 to its consensus sequence on the CD11b promoter and ultimately regulates CD11b expression during the late stages of the neutrophil differentiation of APL-derived promyelocytes. To our knowledge, no previous evidence shows that roscovitine regulates Vav1 activity in myeloid cells, but synthesizing these results with ours suggests roscovitine could be promoting this to drive the phenotypic shift characterizing differentiation.
The adaptor proteins c-Cbl and SLP-76 also promote ATRA-induced differentiation and the G1/G0 arrest of HL-60 cells [11, 12, 42]. Shen and Yen  showed that c-Cbl interacts with CD38 to enhance ATRA-induced differentiation. This is the first report showing that roscovitine enhances the ATRA-induced nuclear enrichment of c-Cbl and SLP-76. Because these adaptor molecules also support signaling by other chemokines that regulate myeloid differentiation, they may also promote signaling pathways collaborating with ATRA during induced differentiation. A well-established collaborating pathway is interferon enhancement of ATRA-driven differentiation . Indeed, we have observed that ATRA induces IRF-1 expression and ectopic expression of IRF-1 propels ATRA-induced differentiation and arrest of these cells . IRF-1 is the well-known transcription factor implicated in being the primary driver of IFN-γ effects . As with adaptor proteins, roscovitine also drives nuclear IRF-1 expression, augmenting ATRA-induced increases that we previously showed enhance Raf/Mek/Erk activation and promote differentiation and cell cycle arrest .
We found that roscovitine enhanced ATRA-induced reduction of cyclin E1, CDK2, pY15 CDK2 and pT160 CDK2, and pS608 RB. Roscovitine enhanced the ATRA-induced increase in p27Kip1 level. The observed ATRA and roscovitine driven reduction in cyclin E1 levels possibly contributes to the following increase in p27Kip1 stability after ATRA-roscovitine treatment because cyclin E-CDK2 complexes can target p27Kip1 for degradation through phosphorylation of Thr187 [65, 66]. RB is the target of CDKs. We found that ATRA induced loss of pS608 RB, which was enhanced by roscovitine. This may have dual effects of enhancing sequestering E2F to cause G1/0 arrest and freeing molecules sequestered by pS608 RB to drive differentiation. RB may thus sequester factors driving differentiation during cell proliferation when S608 is phosphorylated, and sequester factors driving proliferation when S608 phosphorylation is lost and freeing factors that drive differentiation. Hence depending on its phosphorylation state RB may be promoting either proliferation or differentiating by differential sequestration of drivers of these processes.
I tried backing up my Time Machine backup stored on an external USB drive, but Degoo keeps crashing out. Its a 1.4TB file. So it looks like the app is difficult to add terabytes of backup all at once. Not sure if there is a way to add it to degoo. Been trying for days. I might be able to add the files slowly, selecting them one by one, but I was hoping to just add the folder and let Degoo slowly trickle in the files. Will keep trying. 781b155fdc