Biomedical subjects

M J Solomon

Publications and source records attributed to M J Solomon.

At least 19 recordsLinked to original sources

PubMed2007

Ileal pouch-anal anastomosis: the Australasian experience.

OBJECTIVE: The aim of this project was to establish and maintain an internet-based database of all ileal pouch procedures performed in major centres in Australasia. METHOD: The initial three colorectal units contributing data are Auckland, northern Brisbane and Central Sydney Area Health Service. A web-based database was designed. The data collection method was tested on a subgroup of 20 patients to ensure functionality. Data were collected in five main categories: patient demographics, preoperative data, operative details, postoperative complications and functional results. RESULTS: Initial data are presented for 516 patients [363 J, (70%), 133 W (26%), 16 S pouches (3%)]. There were two deaths within 30 days (0.4%). The anastomotic leak rate overall, in handsewn (HSA) and stapled anastomoses (SA) respectively was 5.0%, 8.5% and 3.3% (P=0.02 for difference HSA vs SA). Incidence of pouchitis was 20% (ulcerative colitis 23%, Crohn's disease 20%, indeterminate colitis 22%, familial adenomatous polyposis 9%). Incidence of anal stricture requiring intervention (11% overall) was significantly greater in HSAs than in SAs (16%vs 9%, P=0.02). Incidence of small bowel obstruction at any time postoperatively was 16%. Functional data were available for 234 patients. The median frequency of bowel actions during waking hours was significantly less in W pouches than in J pouches (four vs five, P=0.0005). CONCLUSION: A national web-based database has been developed for access by all Australasian colorectal units. Initial Australasian data compare favourably with other international studies. Pouchitis continues to be a long-term problem. The leak rate and rate of late anal stricture requiring a procedure are higher if the anastomosis is handsewn rather than stapled. Functional results are better with the W pouch than with the J pouch.

Anal Canal↗

PubMed2006

A prepelvic tunnel for the rectus abdominis myocutaneous flap in perineal reconstruction.

BACKGROUND: The use of the transpelvic vertical rectus abdominis myocutaneous (VRAM) flap in pelvic reconstruction is well documented. It can be used to fill large defects after pelvic exenteration, reconstruct the vagina and provide skin coverage in perineal reconstruction. This study examines an alternate prepelvic pathway for the flap to enhance its versatility and reliability. PATIENTS AND METHOD: A female patient with recurrent squamous cell carcinoma in the pelvis, who underwent radical pelvic exenteration and a successful VRAM flap reconstruction with a prepelvic tunnel. RESULTS: The patient experienced a small area of epithelial tip necrosis over the sacral promontory from shear forces. This healed with dressings within two weeks. There were no major flap complications and the patient had good flap integrity at one-year follow-up. The prepelvic pathway for the VRAM flap is advantageous to the conventional transpelvic course in perineal reconstruction. The more direct, shorter path to the defect allows for a more reliable skin paddle design without the need for de-epithelialisation. A greater area of skin paddle is available and creates a more versatile flap with no tension on the pedicle. This is especially in cases where a skin paddle is needed for vaginal reconstruction or when pelvic organs such as bladder and uterus are left in situ. These advantages may result in less flap complications.

Anus Neoplasms↗

PubMed2006

Surgeons' participation in continuing medical education: is it evidence-based?

BACKGROUND: Interactive forms of continuing medical education (CME) are more likely to improve clinical practice than traditional, passive approaches. This study investigated CME participation and preferences among surgeons. METHOD: Questionnaire survey of surgeons in New South Wales, Australia. RESULTS: On average, respondents (n = 418, 77% response rate) committed 364 hours (interquartile range 228-512 hours) to CME per year. Surgeons working at tertiary referral teaching hospitals were twice as likely as those working in other types of hospital to report spending more than 12 hours per month on CME (OR 2.1, 95% CI: 1.4-3.1). Overall, reading accounted for 17% of CME time and attending conferences a further 12%. Clinical audit accounted for significantly less CME time (3.5%) (both P < 0.001). Conferences were considered the single most useful form of CME by 28% (95% CI: 24-33%). Over half (55%, 95% CI: 50-59%) ranked reading as 1 of the 3 most useful types of CME, whereas significantly fewer so ranked clinical audit (6%, 95% CI: 4-9%) (chi2 = 230.8, 1 d.f., P < 0.001). CONCLUSION: Australian surgeons commit a considerable amount of time to CME, but much of this time is spent in passive educational activities. Development of acceptable and effective CME programmes will benefit both surgeons and their patients.

Attitude of Health Personnel↗

PubMed2005

Ano-cutaneous flap repair for complex and recurrent supra-sphincteric anal fistula.

BACKGROUND: Suprasphincteric fistulae remain the most difficult to cure. OBJECTIVES: The purpose of this study was to evaluate the healing rate of suprasphincteric anal fistula treated by ano-cutaneous advancement flap repair, and the impact of this procedure on continence and quality of life. METHOD: Sixteen patients with complex, recurrent or chronic suprasphincteric fistulae associated with significant tissue damage (necrotizing fasciitis, keyhole deformity and anal stenosis) or who had failed previous surgical procedures were treated by ano-cutaneous flap closure. They were assessed pre and postoperatively by the treating surgeon for wound healing and fistula recurrence and later followed up by phone interview using the St Mark's Hospital incontinence score and the Perianal Disease Activity Index (PDAI) as indicators of treatment outcome. RESULTS: Fifteen patients had successful healing of their fistula with the cutaneous flap, with recurrence in only one. The most common short-term complications were minor graft site wound separation, which healed in all cases without intervention, and wound pain, which settled over time and was not associated with recurrence. Continence improved for almost 70% of the patients, with a significant reduction in St Mark's incontinence scores (t = 2.62, 15 d.f., P = 0.02). PDAI also decreased significantly (t = 7.55, 15 d.f., P < 0.001), demonstrating improvement in quality of life for most patients. CONCLUSION: Ano-cutaneous flap can achieve healing of complex and recurrent suprasphincteric anal fistula in patients who had previously failed at other forms of treatment thus improving their quality of life and continence.

Adult↗

PubMed2004

Meta-analysis of short-term outcomes after laparoscopic resection for colorectal cancer.

BACKGROUND: The safety and efficacy of laparoscopic resection (LR) for colorectal cancer remains to be established. METHODS: A meta-analysis of randomized clinical trials comparing the short-term outcomes of laparoscopic with those of open resection for colorectal cancer was undertaken. A literature search was performed for relevant articles published by the end of 2002. Two reviewers independently appraised the trials using a predetermined protocol. Results were analysed using Comprehensive Meta-analysis. RESULTS: The outcomes of 2512 procedures from 12 trials were analysed. LR took on average 32.9 per cent longer to perform than open resection but was associated with lower morbidity rates. Specifically, wound infection rates were significantly lower (odds ratio 0.47 (95 per cent confidence interval 0.28 to 0.80); P = 0.005). In patients undergoing LR, the average time to passage of first flatus was reduced by 33.5 per cent, that to tolerance of a solid diet by 23.9 per cent and that to 80 per cent recovery of peak expiratory flow by 44.3 per cent. Early narcotic analgesia requirements were also reduced by 36.9 per cent, pain at rest by 34.8 per cent and during coughing by 33.9 per cent, and hospital stay by 20.6 per cent. There were no significant differences in perioperative mortality or oncological clearance. CONCLUSION: LR for colorectal cancer is associated with lower morbidity, less pain, a faster recovery and a shorter hospital stay than open resection, without compromising oncological clearance.

C-Reactive Protein↗

PubMed2003

Assessment of the accuracy of transrectal ultrasonography in anorectal neoplasia.

BACKGROUND: Accurate preoperative staging of anorectal neoplasia is required to identify patients for whom local excision or adjuvant therapy may be appropriate. The objectives of this study were to review the accuracy of endoluminal transrectal ultrasonography (TRUS) in the staging of rectal cancers and to determine the learning curve before optimal staging accuracy can be achieved. METHODS: The results of all TRUS examinations for the assessment of anorectal neoplasia performed by two colorectal surgeons at two teaching hospitals of the University of Sydney from 1991 to 2001 were collected prospectively. RESULTS: Of the 433 patients examined by TRUS, 356 were included, of whom 263 (73.9 per cent) had nodal status assessed histologically. Of the 77 patients excluded, 50 had undergone radiotherapy before operation. TRUS achieved excellent accuracy when compared with histopathology reports using kappa statistics for standard Union Internacional Contra la Cancrum (UICC) staging (kappa = 0.89), tumour wall penetration (kappa = 0.70), lymph node detection (kappa = 0.66) and a proposed new staging system (kappa = 0.94). In addition, the increase in TRUS accuracy with operator experience demonstrates the need to perform 50 or more procedures before optimal accuracy is achieved. CONCLUSION: TRUS provides an appropriate investigation with which to select patients with T1 tumours for local excision, and patients with T3 or T4 tumours for preoperative radiotherapy. The relative inaccuracy of staging T2 tumours by TRUS has led to a proposed alternative ultrasonographic staging system.

Anus Neoplasms↗

PubMed2002

Randomized clinical trial of laparoscopic versus open abdominal rectopexy for rectal prolapse.

BACKGROUND: The objectives of this study were to compare both subjective clinical outcomes and the objective stress response of laparoscopic and open abdominal rectopexy in patients with full-thickness rectal prolapse. Abdominal rectopexy for patients with rectal prolapse is well suited for a laparoscopic approach as no resection or anastomosis is necessary. METHODS: Forty patients with a full-thickness rectal prolapse were randomized before operation to a laparoscopic group and an open group. They agreed to conform to a clinical pathway (CP) of liquid diet (CP1) and full mobility (CP2) on day 1, solid diet (CP3) on day 2 and discharge (CP4) before day 5. Their compliance was monitored by an assessor blinded to the operative group, who also rated pain and mobility. Patient-controlled morphine use was documented. Neuroendocrine and immune stress response and respiratory function were measured. RESULTS: Some 75 per cent of all clinical pathway objectives of early recovery were achieved in the laparoscopic group compared with 37 per cent in the open group (P < 0.01). Significant differences in favour of laparoscopy were noted with regard to narcotic requirements, and pain and mobility scores. Differences in objective measures of stress response favouring laparoscopy were found for urinary catecholamines, interleukin 6, serum cortisol and C-reactive protein. No differences were noted in respiratory function but significant respiratory morbidity was greater in the open group (P < 0.05). None of the measured outcomes, subjective or objective, favoured the open group apart from operating time, which was significantly shorter (153 versus 102 min; P < 0.01). CONCLUSION: This study has demonstrated significant subjective and objective differences in favour of a laparoscopic technique for abdominal rectopexy. The advantages were all short term but no evidence of any adverse effect on longer-term outcomes was observed.

Analgesia, Patient-Controlled↗

PubMed2002

p53, DCC and thymidylate synthase as predictors of survival after resection of hepatic metastases from colorectal cancer.

BACKGROUND: Hepatic metastasis from colorectal cancer is a common problem. Hepatic resection offers the only chance of cure. Prognosis of patients following hepatic resection is currently based on clinicopathological factors (of both the primary cancer and the hepatic metastasis), which do not accurately predict the subsequent behaviour of the tumour. The aim of this study was to evaluate three molecular genetic markers - p53, DCC (deleted in colonic cancer) and thymidylate synthase - in both the primary colorectal tumour and the resected hepatic metastases, and to determine their correlation, if any, with survival in patients with resected hepatic metastases from colorectal cancer. METHODS: Sixty-three patients with hepatic metastases and 40 corresponding colorectal primary tumours were studied using immunohistochemical staining for p53, DCC and thymidylate synthase, as well as p53 gene mutations using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) analysis. The results were correlated with survival. RESULTS: There was no correlation between p53, DCC or thymidylate synthase immunohistochemical staining, or between p53 PCR-SSCP analysis, and survival for either hepatic metastases or the colorectal primary tumour. CONCLUSION: Prediction of prognosis in patients having resection of hepatic metastases from colorectal cancer continues to be problematic. Other genetic markers or combination of markers need to be evaluated.

Adult↗

PubMed2001

D box and KEN box motifs in budding yeast Hsl1p are required for APC-mediated degradation and direct binding to Cdc20p and Cdh1p.

The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators. We demonstrated previously that Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC). Here, we investigate regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation. We demonstrate that Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APC(Cdc20) or APC(Cdh1). In coimmunoprecipitation studies, the D box of full-length Hsl1p was critical for association with Cdc20p, whereas the KEN box was important for association with Cdh1p. Fusion of a 206-amino-acid fragment of Hsl1p containing these motifs to a heterologous protein resulted in APC-dependent degradation of the fusion protein that required intact D box and KEN box motifs. Finally, this bacterially expressed Hsl1p fusion protein interacted with Cdc20p and Cdh1p either translated in vitro or expressed in and purified from insect cells. Binding to Cdc20p and Cdh1p was disrupted completely by a D box/KEN box double mutant. These results indicate that D box and KEN box motifs are important for direct binding to the APC machinery, leading to ubiquitination and subsequent protein degradation.

Amino Acid Motifs↗

PubMed2001

Dynamic structure of thermoreversible colloidal gels of adhesive spheres.

The dynamic structure factor f(q,t) of suspensions of adhesive colloidal spheres has been characterized as a function of temperature over the volume fraction range 0.010<phi<0.075. Below a critical temperature that is volume fraction dependent, the suspensions underwent an abrupt, reversible transition in dynamic structure. Below their gel points suspensions became nonergodic, and the time decay of f(q,t) was arrested by as many as five decades. Static light scattering demonstrated that the adhesive spheres formed a fractal cluster gel structure. A recent model of the dynamics of fractal clusters [A. H. Krall and D. A. Weitz, Phys. Rev. Lett. 80, 778 (1998)] was applied to extract the temperature and volume fraction dependence of the characteristic decay times of f(q,t). Immediately above the gel temperature a single stretched exponential decay of f(q,t) was observed. The temperature dependence of the decay time was taualpha approximately epsilon(-1.15+/-0.06), where epsilon=(T-Tgel)/Tgel, and Tgel is the gelation temperature. The argument of the stretched exponential decay p decreased monotonically as the temperature was lowered toward the gel point, until, at gelation, p approximately 0.5. Below the gel temperature, an initial stretched exponential decay of f(q,t) was followed by a plateau. Finally, at long times, an additional exponential decay of the gel f(q,t) was observed. By applying the fractal cluster dynamics model, it was found that the initial decay time, taubeta approximately epsilon(-1.00+/-0.07). The plateau in f(q,t) was due to an upper bound of the mean-squared displacement of gel segments, denoted delta2. The typical magnitude of delta2 was not much greater than the square of the particle radius. The data showed delta2 approximately epsilon(-1.05+/-0.07). The additional exponential decay at long times, taugamma, depended only weakly on epsilon. Its dependence on the scattering vector was taugamma approximately q(-0.53+/-0.06). The argument of the stretched exponential decay of the gel f(q,t) and volume fraction dependence of taubeta and delta2 indicate that the spatial scaling of the gel compliance is consistent with the gel network bonds possessing angular rigidity. The epsilon dependence of the characteristic times taualpha and taubeta could not be fully explained by the fractal cluster dynamics model. The long time decay of f(q,t) exhibited behavior that differed from that recently reported for dilute gels of aqueous colloidal polystyrene [Cipelletti et al., Phys. Rev. Lett. 84, 2275 (2000)]. We hypothesize that the long-time decay in f(q,t) of the gels studied here is due to rare bond disaggregation processes that occur because of the relatively weak interaction between the adhesive spheres (deltaEmin/kT approximately 10) of the thermoreversible gel.

Journal Article↗

PubMed2001

Randomized, controlled trials in surgery: perceived barriers and attitudes of Australian colorectal surgeons.

PURPOSE: Although the randomized, controlled trial has gained preeminence as the criterion standard for evaluating pharmaceutical treatments, randomized controlled trials in surgery have been perceived as difficult to surmount. Furthermore, attitudes of surgeons toward randomized, controlled trials are not well understood. We determined the views of Australian surgeons about feasibility of and barriers to surgical randomized trials. METHODS: All members of the Section of Colon and Rectal Surgery of the Royal Australasian College of Surgeons (n = 147) and all Australian colorectal subspecialist surgeons (n = 72) were mailed a questionnaire that included questions about surgical randomized, controlled trials. RESULTS: A total of 195 surgeons responded (89 percent). Two-thirds (66.7 percent; 95 percent confidence interval 59.5-73.1) of respondents agreed that "Randomized controlled trials should be the study design of choice" to evaluate new surgical procedures. Only 19 percent (95 percent confidence interval 13.9-25.3) endorsed the statement that "too much emphasis is placed on results of randomized controlled trials." Barriers to conducting surgical randomized, controlled trials identified by the majority included insufficient funding (74.4 percent; 95 percent confidence interval 67.5-80.2), a lack of support from the wider surgical community (55.9 percent; 95 percent confidence interval 48.6-62.9), and difficulties in convincing patients to accept random allocation to treatment (62.6 percent; 95 percent confidence interval 55.3-69.3). CONCLUSION: These results reveal positive attitudes among Australian surgeons toward randomized, controlled trials, although concerns about the feasibility of randomized, controlled trials have been reinforced.

Adult↗

PubMed2001

Is there an association between fecal incontinence and lower urinary dysfunction?

BACKGROUND: Urinary and fecal incontinence in females are both common and distressing conditions. Because common pathophysiologic mechanisms have been described, an association between the two would be expected. The aim of this study was to determine whether patients with lower urinary tract dysfunction have concomitant fecal incontinence when compared with age and gender matched community controls and, second, to determine whether they have predisposing factors that have led to lower urinary tract symptoms and concomitant fecal incontinence. METHODS: A case-control study was performed by means of detailed questionnaire and review of investigation results. One thousand consecutive females presenting for urodynamic investigation of lower urinary tract dysfunction, were compared with 148 age and gender matched community controls. RESULTS: Frequent fecal incontinence was significantly more prevalent among all cases than among community controls (5 vs. 0.72 percent, P = 0.023). Occasional fecal incontinence was also more prevalent (24.6 vs. 8.4 percent, P < 0.001). Fecal incontinence was not significantly more prevalent among females with genuine stress incontinence (5.1 percent) when compared with females with detrusor instability (3.8 percent) or any other urodynamic diagnosis. Symptoms of fecal urgency and fecal urge incontinence were significantly more prevalent among those with a urodynamic diagnosis of detrusor instability or sensory urgency than among females with other urodynamic diagnoses or community controls. Multivariate analysis comparing cases with fecal incontinence with other cases and also with community controls did not indicate that individual obstetric factors contributed significantly to the occurrence of fecal incontinence in these patients. CONCLUSIONS: There is an association between genuine stress incontinence, lower urinary tract dysfunction, and symptoms of fecal incontinence, but the exact mechanism of injury related to childbirth trauma is questioned.

Adult↗

PubMed2001

The role of Thr160 phosphorylation of Cdk2 in substrate recognition.

Full activation of cyclin-dependent kinases (Cdks) requires binding to a cyclin and phosphorylation on an activating site equivalent to Thr160 in Cdk2 by the Cdk-activating kinase. Much is known about the effects of cyclin binding, but the role of the activating phosphorylation is less well understood. We have characterized the effects of Thr160 phosphorylation of Cdk2 on its interactions with substrates, particularly with the P + 3 position. We find that an ionic interaction participates in the recognition of the P + 3 position of the substrate and confirms an observation from structural studies indicating that a key element of this recognition is an interaction between the lysine at the P + 3 position and the Thr160 phosphate of Cdk2. The major effect of disrupting the lysine-phosphate interaction was on kcat values rather than Km values, suggesting that the energy from this interaction is used to align the substrate for efficient catalysis. A lack of effect of Thr160 phosphorylation on the ATPase activity of Cdk2 supported this interpretation.

Binding Sites↗

PubMed2001

Island flap perineoplasty for coverage of perineal skin defects after repair of cloacal deformity.

BACKGROUND: Traumatic cloacal defect is an injury sustained during childbirth in which the anovaginal septum is completely disrupted and the anus and vagina open as a common channel. Such injuries result in complete faecal incontinence and are difficult to repair both in terms of improving function and obtaining skin closure. METHODS: Four cases of traumatic cloacal defect with a delayed presentation are illustrated here. All were treated with an overlapping anterior sphincter repair in combination with island flap perineoplasty to achieve skin closure. Anorectal function before and after surgery and the success of achieving primary wound healing were evaluated. RESULTS: In all four cases profound incontinence was found preoperatively; all patients returned to normal or near normal continence following overlapping anterior sphincter repair. Island flap perineoplasty was successful in achieving primary healing in all cases and no flaps were lost to necrosis. CONCLUSIONS: Island flap perineoplasty is an effective method of achieving skin closure after anterior sphincter repair of traumatic cloacal defects.

Adult↗

PubMed2001

Does type of surgeon matter in rectal cancer surgery? Evidence, guideline consensus and surgeons' views.

One of the most obvious but controversial trends in contemporary surgical practice is that of subspecialization. There is a lack of definitive evidence that subspecialization improves cancer outcomes largely because previous research is compromised by confounding variables of referral practice, lack of standardized definitions of surgical skills and selection bias. Randomized controlled trials of generalized versus subspecialist surgical care are unlikely ever to be performed. The present study of surgeons' views about the role of subspecialization in the care of colorectal cancer patients demonstrates partisan reactions among surgeons themselves (89% response rate). Results of national audits will contribute to wider debate about surgical subspecialization in colorectal cancer.

Attitude of Health Personnel↗

PubMed2001

Island flap perineoplasty decreases the incidence of wound breakdown following overlapping anterior sphincter repair.

OBJECTIVE: Overlapping anterior sphincter repair is the accepted treatment for faecal incontinence resulting from sphincter disruption, however, wound breakdown has been reported to occur in up to 30% of patients. The aim of this study was to assess whether the type of wound closure affected the incidence of wound breakdown, and in particular whether island flap perineoplasty decreased this incidence. METHODS: An historical control study was performed evaluating wound outcomes in patients undergoing different methods of wound closure after sphincter repair. Data were obtained from a prospectively collected database. RESULTS: 85 patients who underwent overlapping sphincter repair were studied. Five patients had their wounds left open to heal by granulation. Of the remaining 80 patients, wound dehiscence occurred in 33 patients (41%). When wound breakdown did occur, the mean time to healing was 9.1 weeks. Wound dehiscence was found to occur significantly less frequently in patients having an island flap perineoplasty than in those having other forms of wound closure (15 vs 54%; P=0.0015). The presence of a complex injury such as cloacal defect or recto-vaginal fistula was also found to increase the incidence of wound breakdown, however, performing additional operations at the time of sphincter repair such as levator-plasty, gynaecological procedures or defunctioning colostomy did not affect the incidence of wound disruption. CONCLUSIONS: Wound disruption following overlapping anterior sphincter repair occurs in a significant proportion of patients and results in prolonged healing. Island flap perineoplasty significantly decreases the incidence of wound disruption in comparison to other forms of wound closure.

Journal Article↗

PubMed2001

CAK-independent activation of CDK6 by a viral cyclin.

In normal cells, activation of cyclin-dependent kinases (cdks) requires binding to a cyclin and phosphorylation by the cdk-activating kinase (CAK). The Kaposi's sarcoma-associated herpesvirus encodes a protein with similarity to D-type cyclins. This KSHV-cyclin activates CDK6, alters its substrate specificity, and renders CDK6 insensitive to inhibition by the cdk inhibitor p16(INK4a). Here we investigate the regulation of the CDK6/KSHV-cyclin kinase with the use of purified proteins and a cell-based assay. We find that KSHV-cyclin can activate CDK6 independent of phosphorylation by CAK in vitro. In addition, CAK phosphorylation decreased the p16(INK4a) sensitivity of CDK6/KSHV-cyclin complexes. In cells, expression of CDK6 or to a lesser degree of a nonphosphorylatable CDK6(T177A) together with KSHV-cyclin induced apoptosis, indicating that CDK6 activation by KSHV-cyclin can proceed in the absence of phosphorylation by CAK in vivo. Coexpression of p16 partially protected cells from cell death. p16 and KSHV-cyclin can form a ternary complex with CDK6 that can be detected by binding assays as well as by conformational changes in CDK6. The Kaposi's sarcoma-associated herpesvirus has adopted a clever strategy to render cell cycle progression independent of mitogenic signals, cdk inhibition, or phosphorylation by CAK.

Apoptosis↗

PubMed2000

Dephosphorylation of human cyclin-dependent kinases by protein phosphatase type 2C alpha and beta 2 isoforms.

We previously reported that the activating phosphorylation on cyclin-dependent kinases in yeast (Cdc28p) and in humans (Cdk2) is removed by type 2C protein phosphatases. In this study, we characterize this PP2C-like activity in HeLa cell extract and determine that it is due to PP2C beta 2, a novel PP2C beta isoform, and to PP2C alpha. PP2C alpha and PP2C beta 2 co-purified with Mg(2+)-dependent Cdk2/Cdk6 phosphatase activity in DEAE-Sepharose, Superdex-200, and Mono Q chromatographies. Moreover, purified recombinant PP2C alpha and PP2C beta 2 proteins efficiently dephosphorylated monomeric Cdk2/Cdk6 in vitro. The dephosphorylation of Cdk2 and Cdk6 by PP2C isoforms was inhibited by the binding of cyclins. We found that the PP2C-like activity in HeLa cell extract, partially purified HeLa PP2C alpha and PP2C beta 2 isoforms, and the recombinant PP2Cs exhibited a comparable substrate preference for a phosphothreonine containing substrate, consistent with the conservation of threonine residues at the site of activating phosphorylation in CDKs.

Amino Acid Sequence↗