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Tory MP Aaron Bell, who criticised Boris Johnson in Parliament over lockdown parties, says he has submitted a letter of no confidence in the PM.
Mr Bell made an emotional speech in the Commons on Monday.
During that, he compared the restrictions on his grandmother's funeral in May 2020 with some of the revelations from Sue Gray's report.
She investigated 16 separate gatherings, some of which "should not have been allowed to take place".
These include the 20 May 2020 "bring your own booze" event in the Downing Street garden, which Mr Johnson has apologised for attending.
Posting on Twitter, Mr Bell, MP for Newcastle-under-Lyme, said the "breach of trust" over the No 10 rule breaking and how it had been handled made the PM's position "untenable".
At least 54 MPs are required to write to Sir Graham Brady to trigger a vote on the PM's leadership of the party.
Many of those 17 have cited the PM's participation in parties with staff in No 10 during lockdowns as their motivation to challenge Mr Johnson.
The Metropolitan Police has also launched an investigation into 12 of the parties and a full report by Ms Gray is expected to be published afterwards.
Mr Johnson's spokesman denied he had lost control of Downing Street after a string of close aids quit their jobs.
Analysis, Elizabeth Glinka, Politics Editor, BBC Midlands
On Monday Aaron Bell asked the Prime Minister if he had been a "fool" for following the rules when attending his grandmother's funeral during the first Covid lockdown.
His anger at the prime minister was clear for all to see, and so today's letter cannot be too much of a surprise. Indeed talking to him privately over the last few weeks he's been on the cusp of this many times.
What's important to remember, I think, is that Aaron Bell is not one of the old guard, he's not a disgruntled former minister who held his nose when Boris Johnson was elected leader.
The Newcastle-under-Lyme MP supported Brexit, backed Boris, and won in a red wall seat in 2019.
Whilst not everyone in his local party agrees - indeed some of his nearest neighbours in Stoke-on-Trent are some of the PM's staunchest supporters - it is significant that after taking soundings from his local association and knocking on doors in the constituency this morning he felt the time was now right to go public.
Mr Bell said he had made the decision to submit the letter to Sir Graham after speaking to local councillors and council candidates.
"As someone who backed Brexit and backed Boris Johnson for the leadership in 2019, I am profoundly disappointed that it has come to this," he said.
Addressing Mr Johnson in the House of Commons earlier this week Mr Bell asked Boris Johnson if he "took him for a fool" for abiding by lockdown rules during his grandmother's funeral.
Speaking on Tuesday, Mr Bell said he wanted to register his "disgust" at what had been going on at No 10.
"The reason I didn't go to my nan's house for a cup of tea after was because I felt that those of us who voted for those laws needed to follow them to the letter."
He added he "was standing up for people, people who had much worse experiences" and because "the people who make the rules aren't obeying them".
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